tutorials/05_Create_Parts_With_Overhangs.ipynb

{"cells":[{"cell_type":"markdown","metadata":{"id":"dW2UGT4s32-x"},"source":["# **How to: creating automatic overhangs**\n","\n","How about designing your final plasmid without worrying about each separate part and using a script to add the restriction binding sites, spacer, and overhangs? That’s what you find here!"]},{"cell_type":"markdown","metadata":{"id":"PZJDiu_W3_FT"},"source":["# Configurations for this tutorial\n","\n"]},{"cell_type":"markdown","metadata":{"id":"Xqjf_sf0Am-X"},"source":["\n","First let's run some important settings so you can run this tutorial successfully. \n"]},{"cell_type":"markdown","metadata":{"id":"96PZqanZBAle"},"source":["Colab notebooks use python kernels to run each cell. However, because ***Poly*** is written in **Go language (golang)**, we need to install and configure some things in colab to make feasible run something in go lang here.\n","\n","### **1. In order to start the golang environment, run the line below:**"]},{"cell_type":"code","execution_count":null,"metadata":{"colab":{"base_uri":"https://localhost:8080/"},"executionInfo":{"elapsed":133523,"status":"ok","timestamp":1634727928081,"user":{"displayName":"Isaac Guerreiro","photoUrl":"https://lh3.googleusercontent.com/a-/AOh14Gibo59sdYK-mXxvtGYnmJyGAQyFx3Ml-_Cizj1www=s64","userId":"12892194237443422762"},"user_tz":180},"id":"caT1iMfshw62","outputId":"b54480d9-c7ca-4671-89bb-b2168028739a"},"outputs":[{"name":"stdout","output_type":"stream","text":["\r0% [Working]\r            \rGet:1 http://ppa.launchpad.net/c2d4u.team/c2d4u4.0+/ubuntu bionic InRelease [15.9 kB]\n","\r0% [Waiting for headers] [Connecting to security.ubuntu.com (91.189.91.39)] [Co\r                                                                               \rHit:2 http://archive.ubuntu.com/ubuntu bionic InRelease\n","\r0% [Connecting to security.ubuntu.com (91.189.91.39)] [Connected to cloud.r-pro\r                                                                               \rGet:3 http://archive.ubuntu.com/ubuntu bionic-updates InRelease [88.7 kB]\n","\r                                                                               \rHit:4 http://ppa.launchpad.net/cran/libgit2/ubuntu bionic InRelease\n","\r0% [3 InRelease 50.4 kB/88.7 kB 57%] [Connecting to security.ubuntu.com (91.189\r0% [2 InRelease gpgv 242 kB] [3 InRelease 54.7 kB/88.7 kB 62%] [Connecting to s\r0% [2 InRelease gpgv 242 kB] [Waiting for headers] [Connecting to security.ubun\r                                                                               \rGet:5 https://cloud.r-project.org/bin/linux/ubuntu bionic-cran40/ InRelease [3,626 B]\n","\r0% [2 InRelease gpgv 242 kB] [Waiting for headers] [Connecting to security.ubun\r                                                                               \rGet:6 http://archive.ubuntu.com/ubuntu bionic-backports InRelease [74.6 kB]\n","\r0% [2 InRelease gpgv 242 kB] [6 InRelease 20.0 kB/74.6 kB 27%] [Connecting to s\r                                                                               \rHit:7 http://ppa.launchpad.net/deadsnakes/ppa/ubuntu bionic InRelease\n","\r0% [2 InRelease gpgv 242 kB] [6 InRelease 40.3 kB/74.6 kB 54%] [Connecting to s\r0% [2 InRelease gpgv 242 kB] [Connecting to security.ubuntu.com (91.189.91.39)]\r                                                                               \rHit:8 http://ppa.launchpad.net/graphics-drivers/ppa/ubuntu bionic InRelease\n","\r                                                                               \r0% [2 InRelease gpgv 242 kB] [Waiting for headers] [Waiting for headers]\r                                                                        \rGet:9 http://ppa.launchpad.net/longsleep/golang-backports/ubuntu bionic InRelease [15.4 kB]\n","\r0% [2 InRelease gpgv 242 kB] [Waiting for headers] [9 InRelease 2,572 B/15.4 kB\r                                                                               \r0% [2 InRelease gpgv 242 kB] [Waiting for headers] [Waiting for headers]\r                                                                        \rGet:10 http://security.ubuntu.com/ubuntu bionic-security InRelease [88.7 kB]\n","Ign:11 https://developer.download.nvidia.com/compute/cuda/repos/ubuntu1804/x86_64  InRelease\n","Ign:12 https://developer.download.nvidia.com/compute/machine-learning/repos/ubuntu1804/x86_64  InRelease\n","Hit:13 https://developer.download.nvidia.com/compute/cuda/repos/ubuntu1804/x86_64  Release\n","Hit:14 https://developer.download.nvidia.com/compute/machine-learning/repos/ubuntu1804/x86_64  Release\n","Get:15 http://ppa.launchpad.net/c2d4u.team/c2d4u4.0+/ubuntu bionic/main Sources [1,809 kB]\n","Get:16 http://ppa.launchpad.net/c2d4u.team/c2d4u4.0+/ubuntu bionic/main amd64 Packages [926 kB]\n","Get:17 http://archive.ubuntu.com/ubuntu bionic-updates/universe amd64 Packages [2,213 kB]\n","Get:18 http://archive.ubuntu.com/ubuntu bionic-updates/main amd64 Packages [2,821 kB]\n","Get:19 http://archive.ubuntu.com/ubuntu bionic-updates/restricted amd64 Packages [659 kB]\n","Get:20 https://cloud.r-project.org/bin/linux/ubuntu bionic-cran40/ Packages [69.5 kB]\n","Get:21 http://ppa.launchpad.net/longsleep/golang-backports/ubuntu bionic/main amd64 Packages [6,639 B]\n","Get:24 http://security.ubuntu.com/ubuntu bionic-security/restricted amd64 Packages [614 kB]\n","Get:25 http://security.ubuntu.com/ubuntu bionic-security/main amd64 Packages [2,372 kB]\n","Get:26 http://security.ubuntu.com/ubuntu bionic-security/universe amd64 Packages [1,433 kB]\n","Fetched 13.2 MB in 4s (3,096 kB/s)\n","Reading package lists... 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Done\n","The following additional packages will be installed:\n","  golang-1.17-go golang-1.17-src golang-src\n","Suggested packages:\n","  bzr | brz mercurial subversion\n","The following NEW packages will be installed:\n","  golang-1.17-go golang-1.17-src golang-go golang-src\n","0 upgraded, 4 newly installed, 0 to remove and 63 not upgraded.\n","Need to get 71.8 MB of archives.\n","After this operation, 423 MB of additional disk space will be used.\n","Get:1 http://ppa.launchpad.net/longsleep/golang-backports/ubuntu bionic/main amd64 golang-1.17-src all 1.17.2-1longsleep1+focal [15.2 MB]\n","Get:2 http://ppa.launchpad.net/longsleep/golang-backports/ubuntu bionic/main amd64 golang-1.17-go amd64 1.17.2-1longsleep1+focal [56.6 MB]\n","Get:3 http://ppa.launchpad.net/longsleep/golang-backports/ubuntu bionic/main amd64 golang-src amd64 2:1.17~1longsleep1 [5,056 B]\n","Get:4 http://ppa.launchpad.net/longsleep/golang-backports/ubuntu bionic/main amd64 golang-go amd64 2:1.17~1longsleep1 [24.1 kB]\n","Fetched 71.8 MB in 1s (52.6 MB/s)\n","Selecting previously unselected package golang-1.17-src.\n","(Reading database ... 155047 files and directories currently installed.)\n","Preparing to unpack .../golang-1.17-src_1.17.2-1longsleep1+focal_all.deb ...\n","Unpacking golang-1.17-src (1.17.2-1longsleep1+focal) ...\n","Selecting previously unselected package golang-1.17-go.\n","Preparing to unpack .../golang-1.17-go_1.17.2-1longsleep1+focal_amd64.deb ...\n","Unpacking golang-1.17-go (1.17.2-1longsleep1+focal) ...\n","Selecting previously unselected package golang-src:amd64.\n","Preparing to unpack .../golang-src_2%3a1.17~1longsleep1_amd64.deb ...\n","Unpacking golang-src:amd64 (2:1.17~1longsleep1) ...\n","Selecting previously unselected package golang-go.\n","Preparing to unpack .../golang-go_2%3a1.17~1longsleep1_amd64.deb ...\n","Unpacking golang-go (2:1.17~1longsleep1) ...\n","Setting up golang-1.17-src (1.17.2-1longsleep1+focal) ...\n","Setting up golang-src:amd64 (2:1.17~1longsleep1) ...\n","Setting up golang-1.17-go (1.17.2-1longsleep1+focal) ...\n","Setting up golang-go (2:1.17~1longsleep1) ...\n","Processing triggers for man-db (2.8.3-2ubuntu0.1) ...\n","env: GOPATH=/root/go\n","go: downloading github.com/gopherdata/gophernotes v0.7.3\n","go: downloading github.com/cosmos72/gomacro v0.0.0-20210624153544-b4935e406a41\n","go: downloading github.com/go-zeromq/zmq4 v0.13.0\n","go: downloading github.com/gofrs/uuid v4.0.0+incompatible\n","go: downloading github.com/gofrs/uuid v4.1.0+incompatible\n","go: downloading golang.org/x/xerrors v0.0.0-20200804184101-5ec99f83aff1\n","go: downloading github.com/peterh/liner v1.2.1\n","go: downloading github.com/go-zeromq/goczmq/v4 v4.2.2\n","go: downloading github.com/go-zeromq/goczmq v4.1.0+incompatible\n","go: downloading golang.org/x/sync v0.0.0-20201020160332-67f06af15bc9\n","go: downloading golang.org/x/sync v0.0.0-20210220032951-036812b2e83c\n","go: downloading golang.org/x/tools v0.1.0\n","go: downloading golang.org/x/tools v0.1.7\n","go: downloading github.com/mattn/go-runewidth v0.0.12\n","go: downloading github.com/mattn/go-runewidth v0.0.13\n","go: downloading golang.org/x/sys v0.0.0-20210119212857-b64e53b001e4\n","go: downloading golang.org/x/sys v0.0.0-20211020064051-0ec99a608a1b\n","go: downloading github.com/rivo/uniseg v0.1.0\n","go: downloading github.com/rivo/uniseg v0.2.0\n","go: downloading golang.org/x/mod v0.3.0\n","go: downloading golang.org/x/mod v0.5.1\n","go get: installing executables with 'go get' in module mode is deprecated.\n","\tUse 'go install pkg@version' instead.\n","\tFor more information, see https://golang.org/doc/go-get-install-deprecation\n","\tor run 'go help get' or 'go help install'.\n","\u001b[K\u001b[?25hnpx: installed 1 in 3.24s\n","\u001b[36m> cloned \u001b[1mgopherdata/gophernotes\u001b[22m#\u001b[1mHEAD\u001b[22m to /usr/local/share/jupyter/kernels/gophernotes\u001b[39m\n"]}],"source":["# this process may take a few minutes\n","!add-apt-repository ppa:longsleep/golang-backports -y\n","!apt update\n","!apt install golang-go\n","%env GOPATH=/root/go\n","!go get -u github.com/gopherdata/gophernotes\n","!cp ~/go/bin/gophernotes /usr/bin/\n","!npx degit gopherdata/gophernotes/kernel \\\n","     /usr/local/share/jupyter/kernels/gophernotes"]},{"cell_type":"markdown","metadata":{"id":"cZwRFE9UAGIs"},"source":["### **2. Download important data to run this tutorial**"]},{"cell_type":"code","execution_count":null,"metadata":{"colab":{"base_uri":"https://localhost:8080/"},"executionInfo":{"elapsed":14138,"status":"ok","timestamp":1634727977815,"user":{"displayName":"Isaac Guerreiro","photoUrl":"https://lh3.googleusercontent.com/a-/AOh14Gibo59sdYK-mXxvtGYnmJyGAQyFx3Ml-_Cizj1www=s64","userId":"12892194237443422762"},"user_tz":180},"id":"wHmBJ418_5tX","outputId":"9e247efb-33ca-4105-c6d8-a1806244a6eb"},"outputs":[{"name":"stdout","output_type":"stream","text":["go: downloading github.com/Open-Science-Global/poly v0.11.3\n"]}],"source":["!rm -rf $GOPATH/pkg/mod/github.com/!open-!science-!global\n","!rm -rf $GOPATH/pkg/mod/cache/download/github.com/!open-!science-!global\n","!go get -u github.com/Open-Science-Global/poly@e3e1c61"]},{"cell_type":"code","execution_count":null,"metadata":{"colab":{"base_uri":"https://localhost:8080/"},"executionInfo":{"elapsed":435,"status":"ok","timestamp":1634426318359,"user":{"displayName":"Isaac Guerreiro","photoUrl":"https://lh3.googleusercontent.com/a-/AOh14Gibo59sdYK-mXxvtGYnmJyGAQyFx3Ml-_Cizj1www=s64","userId":"12892194237443422762"},"user_tz":180},"id":"uaxY1xbR-U0F","outputId":"65ab7655-4899-4883-b661-1ce2b85c57db"},"outputs":[{"name":"stdout","output_type":"stream","text":["--2021-10-16 23:18:36--  https://raw.githubusercontent.com/Open-Science-Global/friendzymes-toolkit/main/data/ecoli-k12-cdss.fasta\n","Resolving raw.githubusercontent.com (raw.githubusercontent.com)... 185.199.111.133, 185.199.110.133, 185.199.108.133, ...\n","Connecting to raw.githubusercontent.com (raw.githubusercontent.com)|185.199.111.133|:443... connected.\n","HTTP request sent, awaiting response... 200 OK\n","Length: 5054174 (4.8M) [text/plain]\n","Saving to: ‘ecoli-k12-cdss.fasta’\n","\n","ecoli-k12-cdss.fast 100%[===================>]   4.82M  --.-KB/s    in 0.08s   \n","\n","2021-10-16 23:18:37 (62.6 MB/s) - ‘ecoli-k12-cdss.fasta’ saved [5054174/5054174]\n","\n"]}],"source":["!wget https://raw.githubusercontent.com/Open-Science-Global/friendzymes-cookbook/main/data/create-parts-with-overhangs/pht43-final.gb"]},{"cell_type":"markdown","metadata":{"id":"3STJpf80Bl9Z"},"source":["### **3. Connect Colab Notebook to your GDrive (not required)**"]},{"cell_type":"markdown","metadata":{"id":"ftGzhtH5AOZM"},"source":["The previous code will download the files temporarily. If you want to download them to a folder on your drive and save it for later analysis or if you are already using this notebook to run your own files, you should connect your Google Drive to Colab and you will be able to access, read and save files permanently. \n","So, if you prefer, you can do this with the code line below:\n","\n"]},{"cell_type":"code","execution_count":null,"metadata":{"colab":{"base_uri":"https://localhost:8080/","height":527},"executionInfo":{"elapsed":839877,"status":"error","timestamp":1634427158235,"user":{"displayName":"Isaac Guerreiro","photoUrl":"https://lh3.googleusercontent.com/a-/AOh14Gibo59sdYK-mXxvtGYnmJyGAQyFx3Ml-_Cizj1www=s64","userId":"12892194237443422762"},"user_tz":180},"id":"sh661uGn_8R1","outputId":"9d874164-d5ca-4b63-ac83-aa544d2d0fdd"},"outputs":[{"ename":"KeyboardInterrupt","evalue":"ignored","output_type":"error","traceback":["\u001b[0;31m---------------------------------------------------------------------------\u001b[0m","\u001b[0;31mKeyboardInterrupt\u001b[0m                         Traceback (most recent call last)","\u001b[0;32m/usr/local/lib/python3.7/dist-packages/ipykernel/kernelbase.py\u001b[0m in \u001b[0;36m_input_request\u001b[0;34m(self, prompt, ident, parent, password)\u001b[0m\n\u001b[1;32m    728\u001b[0m             \u001b[0;32mtry\u001b[0m\u001b[0;34m:\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n\u001b[0;32m--> 729\u001b[0;31m                 \u001b[0mident\u001b[0m\u001b[0;34m,\u001b[0m \u001b[0mreply\u001b[0m \u001b[0;34m=\u001b[0m \u001b[0mself\u001b[0m\u001b[0;34m.\u001b[0m\u001b[0msession\u001b[0m\u001b[0;34m.\u001b[0m\u001b[0mrecv\u001b[0m\u001b[0;34m(\u001b[0m\u001b[0mself\u001b[0m\u001b[0;34m.\u001b[0m\u001b[0mstdin_socket\u001b[0m\u001b[0;34m,\u001b[0m \u001b[0;36m0\u001b[0m\u001b[0;34m)\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n\u001b[0m\u001b[1;32m    730\u001b[0m             \u001b[0;32mexcept\u001b[0m \u001b[0mException\u001b[0m\u001b[0;34m:\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n","\u001b[0;32m/usr/local/lib/python3.7/dist-packages/jupyter_client/session.py\u001b[0m in \u001b[0;36mrecv\u001b[0;34m(self, socket, mode, content, copy)\u001b[0m\n\u001b[1;32m    802\u001b[0m         \u001b[0;32mtry\u001b[0m\u001b[0;34m:\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n\u001b[0;32m--> 803\u001b[0;31m             \u001b[0mmsg_list\u001b[0m \u001b[0;34m=\u001b[0m \u001b[0msocket\u001b[0m\u001b[0;34m.\u001b[0m\u001b[0mrecv_multipart\u001b[0m\u001b[0;34m(\u001b[0m\u001b[0mmode\u001b[0m\u001b[0;34m,\u001b[0m \u001b[0mcopy\u001b[0m\u001b[0;34m=\u001b[0m\u001b[0mcopy\u001b[0m\u001b[0;34m)\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n\u001b[0m\u001b[1;32m    804\u001b[0m         \u001b[0;32mexcept\u001b[0m \u001b[0mzmq\u001b[0m\u001b[0;34m.\u001b[0m\u001b[0mZMQError\u001b[0m \u001b[0;32mas\u001b[0m \u001b[0me\u001b[0m\u001b[0;34m:\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n","\u001b[0;32m/usr/local/lib/python3.7/dist-packages/zmq/sugar/socket.py\u001b[0m in \u001b[0;36mrecv_multipart\u001b[0;34m(self, flags, copy, track)\u001b[0m\n\u001b[1;32m    624\u001b[0m         \"\"\"\n\u001b[0;32m--> 625\u001b[0;31m         \u001b[0mparts\u001b[0m \u001b[0;34m=\u001b[0m \u001b[0;34m[\u001b[0m\u001b[0mself\u001b[0m\u001b[0;34m.\u001b[0m\u001b[0mrecv\u001b[0m\u001b[0;34m(\u001b[0m\u001b[0mflags\u001b[0m\u001b[0;34m,\u001b[0m \u001b[0mcopy\u001b[0m\u001b[0;34m=\u001b[0m\u001b[0mcopy\u001b[0m\u001b[0;34m,\u001b[0m \u001b[0mtrack\u001b[0m\u001b[0;34m=\u001b[0m\u001b[0mtrack\u001b[0m\u001b[0;34m)\u001b[0m\u001b[0;34m]\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n\u001b[0m\u001b[1;32m    626\u001b[0m         \u001b[0;31m# have first part already, only loop while more to receive\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n","\u001b[0;32mzmq/backend/cython/socket.pyx\u001b[0m in \u001b[0;36mzmq.backend.cython.socket.Socket.recv\u001b[0;34m()\u001b[0m\n","\u001b[0;32mzmq/backend/cython/socket.pyx\u001b[0m in \u001b[0;36mzmq.backend.cython.socket.Socket.recv\u001b[0;34m()\u001b[0m\n","\u001b[0;32mzmq/backend/cython/socket.pyx\u001b[0m in \u001b[0;36mzmq.backend.cython.socket._recv_copy\u001b[0;34m()\u001b[0m\n","\u001b[0;32m/usr/local/lib/python3.7/dist-packages/zmq/backend/cython/checkrc.pxd\u001b[0m in \u001b[0;36mzmq.backend.cython.checkrc._check_rc\u001b[0;34m()\u001b[0m\n","\u001b[0;31mKeyboardInterrupt\u001b[0m: ","\nDuring handling of the above exception, another exception occurred:\n","\u001b[0;31mKeyboardInterrupt\u001b[0m                         Traceback (most recent call last)","\u001b[0;32m<ipython-input-4-d5df0069828e>\u001b[0m in \u001b[0;36m<module>\u001b[0;34m()\u001b[0m\n\u001b[1;32m      1\u001b[0m \u001b[0;32mfrom\u001b[0m \u001b[0mgoogle\u001b[0m\u001b[0;34m.\u001b[0m\u001b[0mcolab\u001b[0m \u001b[0;32mimport\u001b[0m \u001b[0mdrive\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n\u001b[0;32m----> 2\u001b[0;31m \u001b[0mdrive\u001b[0m\u001b[0;34m.\u001b[0m\u001b[0mmount\u001b[0m\u001b[0;34m(\u001b[0m\u001b[0;34m'/content/drive'\u001b[0m\u001b[0;34m)\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n\u001b[0m","\u001b[0;32m/usr/local/lib/python3.7/dist-packages/google/colab/drive.py\u001b[0m in \u001b[0;36mmount\u001b[0;34m(mountpoint, force_remount, timeout_ms, use_metadata_server)\u001b[0m\n\u001b[1;32m    111\u001b[0m       \u001b[0mtimeout_ms\u001b[0m\u001b[0;34m=\u001b[0m\u001b[0mtimeout_ms\u001b[0m\u001b[0;34m,\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n\u001b[1;32m    112\u001b[0m       \u001b[0muse_metadata_server\u001b[0m\u001b[0;34m=\u001b[0m\u001b[0muse_metadata_server\u001b[0m\u001b[0;34m,\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n\u001b[0;32m--> 113\u001b[0;31m       ephemeral=ephemeral)\n\u001b[0m\u001b[1;32m    114\u001b[0m \u001b[0;34m\u001b[0m\u001b[0m\n\u001b[1;32m    115\u001b[0m \u001b[0;34m\u001b[0m\u001b[0m\n","\u001b[0;32m/usr/local/lib/python3.7/dist-packages/google/colab/drive.py\u001b[0m in \u001b[0;36m_mount\u001b[0;34m(mountpoint, force_remount, timeout_ms, use_metadata_server, ephemeral)\u001b[0m\n\u001b[1;32m    290\u001b[0m       \u001b[0;32mwith\u001b[0m \u001b[0m_output\u001b[0m\u001b[0;34m.\u001b[0m\u001b[0muse_tags\u001b[0m\u001b[0;34m(\u001b[0m\u001b[0;34m'dfs-auth-dance'\u001b[0m\u001b[0;34m)\u001b[0m\u001b[0;34m:\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n\u001b[1;32m    291\u001b[0m         \u001b[0;32mwith\u001b[0m \u001b[0mopen\u001b[0m\u001b[0;34m(\u001b[0m\u001b[0mfifo\u001b[0m\u001b[0;34m,\u001b[0m \u001b[0;34m'w'\u001b[0m\u001b[0;34m)\u001b[0m \u001b[0;32mas\u001b[0m \u001b[0mfifo_file\u001b[0m\u001b[0;34m:\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n\u001b[0;32m--> 292\u001b[0;31m           \u001b[0mfifo_file\u001b[0m\u001b[0;34m.\u001b[0m\u001b[0mwrite\u001b[0m\u001b[0;34m(\u001b[0m\u001b[0mget_code\u001b[0m\u001b[0;34m(\u001b[0m\u001b[0mauth_prompt\u001b[0m\u001b[0;34m)\u001b[0m \u001b[0;34m+\u001b[0m \u001b[0;34m'\\n'\u001b[0m\u001b[0;34m)\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n\u001b[0m\u001b[1;32m    293\u001b[0m       \u001b[0mwrote_to_fifo\u001b[0m \u001b[0;34m=\u001b[0m \u001b[0;32mTrue\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n\u001b[1;32m    294\u001b[0m     \u001b[0;32melif\u001b[0m \u001b[0mcase\u001b[0m \u001b[0;34m==\u001b[0m \u001b[0;36m5\u001b[0m\u001b[0;34m:\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n","\u001b[0;32m/usr/local/lib/python3.7/dist-packages/ipykernel/kernelbase.py\u001b[0m in \u001b[0;36mraw_input\u001b[0;34m(self, prompt)\u001b[0m\n\u001b[1;32m    702\u001b[0m             \u001b[0mself\u001b[0m\u001b[0;34m.\u001b[0m\u001b[0m_parent_ident\u001b[0m\u001b[0;34m,\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n\u001b[1;32m    703\u001b[0m             \u001b[0mself\u001b[0m\u001b[0;34m.\u001b[0m\u001b[0m_parent_header\u001b[0m\u001b[0;34m,\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n\u001b[0;32m--> 704\u001b[0;31m             \u001b[0mpassword\u001b[0m\u001b[0;34m=\u001b[0m\u001b[0;32mFalse\u001b[0m\u001b[0;34m,\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n\u001b[0m\u001b[1;32m    705\u001b[0m         )\n\u001b[1;32m    706\u001b[0m \u001b[0;34m\u001b[0m\u001b[0m\n","\u001b[0;32m/usr/local/lib/python3.7/dist-packages/ipykernel/kernelbase.py\u001b[0m in \u001b[0;36m_input_request\u001b[0;34m(self, prompt, ident, parent, password)\u001b[0m\n\u001b[1;32m    732\u001b[0m             \u001b[0;32mexcept\u001b[0m \u001b[0mKeyboardInterrupt\u001b[0m\u001b[0;34m:\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n\u001b[1;32m    733\u001b[0m                 \u001b[0;31m# re-raise KeyboardInterrupt, to truncate traceback\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n\u001b[0;32m--> 734\u001b[0;31m                 \u001b[0;32mraise\u001b[0m \u001b[0mKeyboardInterrupt\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n\u001b[0m\u001b[1;32m    735\u001b[0m             \u001b[0;32melse\u001b[0m\u001b[0;34m:\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n\u001b[1;32m    736\u001b[0m                 \u001b[0;32mbreak\u001b[0m\u001b[0;34m\u001b[0m\u001b[0;34m\u001b[0m\u001b[0m\n","\u001b[0;31mKeyboardInterrupt\u001b[0m: "]}],"source":["from google.colab import drive\n","drive.mount('/content/drive')"]},{"cell_type":"markdown","metadata":{"id":"GbPLWTJ05uH4"},"source":["## After running these steps, click on **Runtime** in the menu bar & **Change Runtime Type** to Go, if it hasn't changed yet.\n","\n","This will make Colab starting use a open source go kernel called Gopher Notes.\n","\n","Now we are ready to work."]},{"cell_type":"markdown","metadata":{"id":"flsk3_roXwdM"},"source":["# **Creating automatic overhangs**"]},{"cell_type":"markdown","metadata":{"id":"34gduXzXl-Nb"},"source":["## **Importing packages and pre-requisites**"]},{"cell_type":"code","execution_count":null,"metadata":{"id":"gQVnSh-tmEvz"},"outputs":[],"source":["package main\n","\n","import (\n","\t\"fmt\"\n","\t\"log\"\n","\t\"os\"\n","\t\"path/filepath\"\n","\t\"strings\"\n","\t\"strconv\"\n","\t\"sync\"\n","\t\"math/rand\"\n","\n","  \"github.com/Open-Science-Global/poly\"\n","\t\"github.com/Open-Science-Global/poly/checks\"\n","\t\"github.com/Open-Science-Global/poly/io/fasta\"\n","\t\"github.com/Open-Science-Global/poly/io/genbank\"\n","\t\"github.com/Open-Science-Global/poly/linearfold\"\n","\t\"github.com/Open-Science-Global/poly/synthesis\"\n","\t\"github.com/Open-Science-Global/poly/transform\"\n","\t\"github.com/Open-Science-Global/poly/transform/codon\"\n","\t\"github.com/Open-Science-Global/poly/finder\"\n",")"]},{"cell_type":"markdown","metadata":{"id":"JjOp36MzMUfw"},"source":["## **1. Preparations for creating automatic overhangs: functions set up**\n"]},{"cell_type":"markdown","metadata":{"id":"wbkdF0VqWTUg"},"source":["Run the cells below to generate the function to automatic create based on the sequences of your choice. \n","For example: You created your final plasmid with everything you think will be necessary to get from the organism your final objective, but would be nice to have a tool to separate all your project in parts, already organized by type using the assembly system connectors from your favorite assembly standard. In this case is the Friendzymes extended MoClo standard, but feel free to change to your pattern.\n","\n","\n","**Important points:**\n","*   We have below ONE main function (AutomaticAnnotation) and 15 subfunctions (randonDnaSequence, addBbsiStructureFoward, addBbsiStructureReverse, addOverhangs, reateListPromoters, createListTerminator, createListFlankReverse, createListTargetSelection, createListEcoliSelectionOrigin, createListEcoliOrigin, createListRbs, createListEcoliSelection, createListCds, filterByType, generateOverhangs) that are inside the main one and also need to be set. \n","*   Overall, you don't need to change anything to save and use these functions in the second step of this tutorial (2. Running), except if you want to use a different assembly standard, so we highly recommend you to understand line bve line and make the necessary changes."]},{"cell_type":"code","execution_count":null,"metadata":{"colab":{"base_uri":"https://localhost:8080/"},"executionInfo":{"elapsed":618,"status":"ok","timestamp":1634731219486,"user":{"displayName":"Isaac Guerreiro","photoUrl":"https://lh3.googleusercontent.com/a-/AOh14Gibo59sdYK-mXxvtGYnmJyGAQyFx3Ml-_Cizj1www=s64","userId":"12892194237443422762"},"user_tz":180},"id":"M4a0RTRKEzhv","outputId":"372b3ea5-a727-468e-9873-7d8f404e7a98"},"outputs":[{"data":{"text/plain":["CGTCCCTGAT"]},"execution_count":3,"metadata":{},"output_type":"execute_result"}],"source":["// This functions generates a random sequence of length N using a specific seed.\n","func randomDnaSequence(length int, seed int64) string {\n","  var dnaAlphabet = []rune(\"ATCG\")\n","\trand.Seed(seed)\n","\n","\trandomSequence := make([]rune, length)\n","\n","\tfor basepair := range randomSequence {\n","\t\t  randomIndex := rand.Intn(len(dnaAlphabet))\n","\t\t\trandomSequence[basepair] = dnaAlphabet[randomIndex]\n","\t}\n","\treturn string(randomSequence)\n","}\n","\n","randomDnaSequence(10, int64(rand.Intn(100)))"]},{"cell_type":"code","execution_count":null,"metadata":{"id":"ycykE9kxfmuj"},"outputs":[],"source":["// 15 random bp -> bbsi cut site forward GAAGAC -> 2bp -> bbsi overhang GGAG -> random 8bp ->\n","// bsai site forward -> bsai overhang -> main sequence -> bsai overhang 2 -> bsai site reverse -> random 8bp ->\n","// bbsi overhang CGCT -> 2bp -> bbsi cute site reverse GTCTTC -> 15 random bp\n","func addBbsiStructureFoward(internalOverhang string) string {\n","  bbsiFoward := \"GAAGAC\" \n","  bbsiOverhangFoward := \"GGAG\"\n","  randomFoward := randomDnaSequence(15, int64(rand.Intn(10000)))\n","  twoRandomFoward := randomDnaSequence(2, int64(rand.Intn(10000))) \n","  eightRandomFoward := randomDnaSequence(8, int64(rand.Intn(10000))) \n","\n","  return randomFoward  + bbsiFoward + twoRandomFoward + bbsiOverhangFoward + eightRandomFoward + internalOverhang\n","\n","}\n","\n","func addBbsiStructureReverse(internalOverhang string) string {\n","  bbsiReverse := \"GTCTTC\"\n","  bbsiOverhangReverse := \"CGCT\"\n","  randomReverse := randomDnaSequence(15, int64(rand.Intn(10000)))\n","  twoRandomReverse := randomDnaSequence(2, int64(rand.Intn(10000)))\n","  eightRandomReverse := randomDnaSequence(8, int64(rand.Intn(10000)))\n","\n","  return internalOverhang + eightRandomReverse + bbsiOverhangReverse + twoRandomReverse + bbsiReverse + randomReverse\n","\n","}"]},{"cell_type":"markdown","metadata":{"id":"2ZOIxcdFdQ4w"},"source":["The function below *addOverhangs* is responsible for receiving a full sequence and a list of annotations (called features as genbank standard) and extract from the final sequence a specific part (a promotor for example) and add the correct overhangs for a specific standard."]},{"cell_type":"code","execution_count":null,"metadata":{"id":"ydxusbFQh-fB"},"outputs":[],"source":["type MoCloPart struct {\n","  sequence string\n","  name string\n","  typePart string\n","}\n","\n","//Features\n","func addOverhangs(sequence string, features []poly.Feature, fiveOverhang string, threeOverhang string) []MoCloPart {\n","  var parts []MoCloPart\n","  for _, feature := range features {\n","    annotationSequence := sequence[feature.SequenceLocation.Start:feature.SequenceLocation.End]\n","    parts = append(parts, MoCloPart{\n","                            strings.ToUpper(fiveOverhang + annotationSequence + threeOverhang),\n","                            feature.Attributes[\"label\"],\n","                            feature.Type,\n","                          })\n","  }\n","  return parts\n","}"]},{"cell_type":"markdown","metadata":{"id":"IidofEUxegBZ"},"source":["We also created functions for each specific part type to add overhangs following our specific assembly standard."]},{"cell_type":"code","execution_count":null,"metadata":{"id":"KXlVgYz7iFrF"},"outputs":[],"source":["import \"github.com/Open-Science-Global/poly\"\n","\n","func createListPromoters(sequence string, features []poly.Feature) []MoCloPart {\n","  spacer := \"T\"\n","  bsaiFoward := \"GGTCTC\" + spacer\n","  bsaiReverse := spacer + \"GAGACC\"\n","  fiveOverhang := bsaiFoward + \"GGAG\"\n","  threeOverhang := \"TACT\" + bsaiReverse\n","  return addOverhangs(sequence, features, addBbsiStructureFoward(fiveOverhang), addBbsiStructureReverse(threeOverhang))\n","}\n","\n","// An observation it's when we have a promoter it's close to an insulator, or a promoter following\n","// a recombination site we should use other overhangs, and we have to create another type annotations\n","// instead of PR\n","\n","func createListRbs(sequence string, features []poly.Feature) []MoCloPart {\n","  spacer := \"T\"\n","  bsaiFoward := \"GGTCTC\" + spacer\n","  bsaiReverse := spacer + \"GAGACC\"\n","  fiveOverhang := bsaiFoward + \"TACT\"\n","  threeOverhang := \"GACC\" + bsaiReverse\n","  return addOverhangs(sequence, features, addBbsiStructureFoward(fiveOverhang), addBbsiStructureReverse(threeOverhang))\n","}\n","\n","func createListCds(sequence string, features []poly.Feature) []MoCloPart {\n","  spacer := \"T\"\n","  bsaiFoward := \"GGTCTC\" + spacer\n","  bsaiReverse := spacer + \"GAGACC\"\n","  fiveOverhang := bsaiFoward + \"AATG\"\n","  threeOverhang := \"GCTT\" + bsaiReverse\n","  return addOverhangs(sequence, features, addBbsiStructureFoward(fiveOverhang), addBbsiStructureReverse(threeOverhang))\n","}\n","\n","// if we have a CDS close to a c terminal tag we should use another overhangs\n","// and another type annotations instead of CS \n","\n","func createListTerminator(sequence string, features []poly.Feature) []MoCloPart {\n","  spacer := \"T\"\n","  bsaiFoward := \"GGTCTC\" + spacer\n","  bsaiReverse := spacer + \"GAGACC\"\n","  fiveOverhang := bsaiFoward + \"GCTT\"\n","  threeOverhang := \"CGCT\" + bsaiReverse\n","  return addOverhangs(sequence, features, addBbsiStructureFoward(fiveOverhang), addBbsiStructureReverse(threeOverhang))\n","}\n","\n","func createListTargetSelection(sequence string, features []poly.Feature) []MoCloPart {\n","  spacer := \"T\"\n","  bsaiFoward := \"GGTCTC\" + spacer\n","  bsaiReverse := spacer + \"GAGACC\"\n","  fiveOverhang := bsaiFoward + \"AAGG\"\n","  threeOverhang := \"ATGA\" + bsaiReverse\n","  return addOverhangs(sequence, features, addBbsiStructureFoward(fiveOverhang), addBbsiStructureReverse(threeOverhang))\n","}\n","\n","func createListEcoliSelection(sequence string, features []poly.Feature) []MoCloPart {\n","  spacer := \"T\"\n","  bsaiFoward := \"GGTCTC\" + spacer\n","  bsaiReverse := spacer + \"GAGACC\"\n","  fiveOverhang := bsaiFoward + \"GCAA\"\n","  threeOverhang := \"ACTA\" + bsaiReverse\n","  return addOverhangs(sequence, features, addBbsiStructureFoward(fiveOverhang), addBbsiStructureReverse(threeOverhang))\n","}\n","\n","func createListEcoliOrigin(sequence string, features []poly.Feature) []MoCloPart {\n","  spacer := \"T\"\n","  bsaiFoward := \"GGTCTC\" + spacer\n","  bsaiReverse := spacer + \"GAGACC\"\n","  fiveOverhang := bsaiFoward + \"ACTA\"\n","  threeOverhang := \"AAAA\" + bsaiReverse\n","  return addOverhangs(sequence, features, addBbsiStructureFoward(fiveOverhang), addBbsiStructureReverse(threeOverhang))\n","}\n","\n","func createListFlankReverse(sequence string, features []poly.Feature) []MoCloPart {\n","  spacer := \"T\"\n","  bsaiFoward := \"GGTCTC\" + spacer\n","  bsaiReverse := spacer + \"GAGACC\"\n","  fiveOverhang := bsaiFoward + \"AAAA\"\n","  threeOverhang := \"AAGG\" + bsaiReverse\n","  return addOverhangs(sequence, features, addBbsiStructureFoward(fiveOverhang), addBbsiStructureReverse(threeOverhang))\n","}\n","\n","func createListEcoliSelectionOrigin(sequence string, features []poly.Feature) []MoCloPart {\n","  spacer := \"T\"\n","  bsaiFoward := \"GGTCTC\" + spacer\n","  bsaiReverse := spacer + \"GAGACC\"\n","  fiveOverhang := bsaiFoward + \"GCAA\"\n","  threeOverhang := \"AAAA\" + bsaiReverse\n","  return addOverhangs(sequence, features, addBbsiStructureFoward(fiveOverhang), addBbsiStructureReverse(threeOverhang))\n","}"]},{"cell_type":"markdown","metadata":{"id":"lqi3rh-Kfw1I"},"source":["The *filterByType* is a function responsible for filtering annotation by specific metadata (the Type property in the feature object), so we could automatic identify annotation by their part types and correctly add the overhangs."]},{"cell_type":"code","execution_count":null,"metadata":{"id":"tYzcxSZnhKFy"},"outputs":[],"source":["import \"strings\"\n","\n","func filterByType(features []poly.Feature, partType string) []poly.Feature {\n","  var list []poly.Feature\n","  for _, feature := range features {\n","    if strings.ToUpper(feature.Type) == partType {\n","      list = append(list, feature)\n","    }\n","  }\n","  return list\n","}"]},{"cell_type":"markdown","metadata":{"id":"QhctodbGieE5"},"source":["The *generateOverhangs* function basically receives a specific tag and generate all the overhangs for type of part."]},{"cell_type":"code","execution_count":null,"metadata":{"id":"FWQIA6OgokMa"},"outputs":[],"source":["import \"log\"\n","\n","func generateOverhangs(tag string, sequence string, features []poly.Feature) []MoCloPart {\n","\n","  switch tag {\n","    case \"PR\": {\n","        return createListPromoters(sequence, features)\n","    }\n","    case \"TS\": {\n","        return createListTerminator(sequence, features)\n","    }\n","    case \"FR\": {\n","        return createListFlankReverse(sequence, features)\n","    }\n","    case \"ES\": {\n","        return createListEcoliSelection(sequence, features)\n","    }\n","    case \"EO\": {\n","        return createListEcoliOrigin(sequence, features)\n","    }\n","    case \"RB\": {\n","        return createListRbs(sequence, features)\n","    }\n","    case \"ES+EO\": {\n","        return createListEcoliSelectionOrigin(sequence, features)\n","    }\n","  }\n","    log.Fatal(\"Pass a tag of a possible part category!\")\n","}"]},{"cell_type":"code","execution_count":null,"metadata":{"id":"ZW4AeCW1jtQB"},"outputs":[],"source":["func AutomaticOverhangs(sequence poly.Sequence) []MoCloPart {\n","  tags := []string{\"PR\", \"TS\", \"FR\", \"ES\", \"EO\", \"RB\", \"ES+EO\"}\n","\n","  var allParts []MoCloPart\n","  for _, tag := range tags {\n","    filteredParts := filterByType(sequence.Features, tag)\n","    if (len(filteredParts) > 0) {\n","      parts := generateOverhangs(tag, sequence.Sequence, filteredParts)\n","      allParts = append(allParts, parts...)\n","    }\n","  }\n","  return allParts\n","}"]},{"cell_type":"markdown","metadata":{"id":"i-iiuQq4i6yT"},"source":["## **2. Running**\n"]},{"cell_type":"markdown","metadata":{"id":"iEs3PFEhjFtq"},"source":["Now that we have all the functions necessary, lets combine everything together by receiving a final plasmid sequence the we called *pht-43-final* sequence."]},{"cell_type":"code","execution_count":null,"metadata":{"id":"RjOjkqru0Ak-"},"outputs":[],"source":["// For this tutorial, we have already downloaded this in the settings part (in \"Download important data to run this tutorial\")\n","pht43 := genbank.Read(\"./pht43-final.gb\")"]},{"cell_type":"markdown","metadata":{"id":"vzuOkV1QlDmY"},"source":["Run the AutomaticAnnotation funtion with the final plasmid and take a look, you received all the annotations with Type properties that matches the tags created in the *generatedOverhangs* function with the overhangs already created. "]},{"cell_type":"code","execution_count":null,"metadata":{"id":"FmbtF6hNjoKp"},"outputs":[],"source":["parts := AutomaticOverhangs(pht43)"]},{"cell_type":"markdown","metadata":{"id":"8b8vaOPCmJD4"},"source":["Now transform all this sequences in Fasta objects to create a Fasta file with all your sequences already in you Assembly Standard!"]},{"cell_type":"code","execution_count":null,"metadata":{"id":"MdGCZe5BvNLU"},"outputs":[],"source":["var fastas []fasta.Fasta\n","\n","for _, part := range parts {\n","  fastas = append(fastas, fasta.Fasta{part.name + \"|\" + part.typePart, part.sequence})\n","}\n","\n","fasta.Write(fastas, \"./allPartsWithOverhangs.fasta\")"]}],"metadata":{"colab":{"collapsed_sections":["96PZqanZBAle","3STJpf80Bl9Z"],"name":"#5 - CreatePartsWithOverhangs.ipynb","provenance":[{"file_id":"1NE42_1QHeAyEGTGuAxpsgtGsyJIcIr3I","timestamp":1634430571139},{"file_id":"1JVQfcSFEBoIV4TNCDRYCkcLssQhfp2S2","timestamp":1634426177979},{"file_id":"1GWFFYUcduoykRm08R6Jq7OacGQmDF1BS","timestamp":1634327217511},{"file_id":"1Ts_naBl0P_KfVvwC90Fb-JGWDeP6qjrV","timestamp":1634234790466},{"file_id":"14Bh2CljYULhD03AKCNGlJ3YgM9T5mcRa","timestamp":1627231003334},{"file_id":"1fZTAiBlR5eicaMu4nhhtfeJZEGMUAMHD","timestamp":1626732496856},{"file_id":"1NGDMQi-OGV5-XAbOgz87JWtSc4l2hHyh","timestamp":1626622594804}]},"kernelspec":{"display_name":"Go","name":"gophernotes"}},"nbformat":4,"nbformat_minor":0}