2019 molecular biology

New plasmid

Addgene pICH47732

MoClo level 1 destination vector pL1F-1

gen bank entry

from Bartlett lab

amp-r

Lac-Z cassette

Test for restriction sites:

per 20 uL reaction:

• 10 uL midiprep DNA (40 ng/uL)

• 2 uL 10x NEB cut smart

• 1 uL enzyme (all from NEB) or water

• 7 uL water

  • water/control
  • BamH1
  • EcoR1
  • Kpn

• incubate 1 hr at 37

• Mix with 4 uL loading dye

• Run in 1% agarose gel ~1.5 hr (needs a long run - the DNA pieces are long)

• See attached picture below

Sequence

    1 gtggggccca ctgcatccac cccagtacat taaaaacgtc cgcaatgtgt tattaagttg
   61 tctaagcgtc aatttgttta caccacaata tatcctgcca ccagccagcc aacagctccc
  121 cgaccggcag ctcggcacaa aatcaccact cgatacaggc agcccatcag tcagatcagg
  181 atctcctttg cgacgctcac cgggctggtt gccctcgccg ctgggctggc ggccgtctat
  241 ggccctgcaa acgcgccaga aacgccgtcg aagccgtgtg cgagacaccg cggccgccgg
  301 cgttgtggat acctcgcgga aaacttggcc ctcactgaca gatgaggggc ggacgttgac
  361 acttgagggg ccgactcacc cggcgcggcg ttgacagatg aggggcaggc tcgatttcgg
  421 ccggcgacgt ggagctggcc agcctcgcaa atcggcgaaa acgcctgatt ttacgcgagt
  481 ttcccacaga tgatgtggac aagcctgggg ataagtgccc tgcggtattg acacttgagg
  541 ggcgcgacta ctgacagatg aggggcgcga tccttgacac ttgaggggca gagtgctgac
  601 agatgagggg cgcacctatt gacatttgag gggctgtcca caggcagaaa atccagcatt
  661 tgcaagggtt tccgcccgtt tttcggccac cgctaacctg tcttttaacc tgcttttaaa
  721 ccaatattta taaaccttgt ttttaaccag ggctgcgccc tgtgcgcgtg accgcgcacg
  781 ccgaaggggg gtgccccccc ttctcgaacc ctcccggccc gctaacgcgg gcctcccatc
  841 cccccagggg ctgcgcccct cggccgcgaa cggcctcacc ccaaaaatgg cagcgctggc
  901 caattcgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
  961 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
 1021 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
 1081 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
 1141 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
 1201 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
 1261 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
 1321 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
 1381 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
 1441 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
 1501 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
 1561 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
 1621 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
 1681 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg ttctcgcggt
 1741 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
 1801 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
 1861 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
 1921 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
 1981 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
 2041 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
 2101 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
 2161 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
 2221 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
 2281 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
 2341 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
 2401 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
 2461 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
 2521 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
 2581 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
 2641 agcaacgcgg cctttttacg gttcctggca gatcctagat gtggcgcaac gatgccggcg
 2701 acaagcagga gcgcaccgac ttcttccgca tcaagtgttt tggctctcag gccgaggccc
 2761 acggcaagta tttgggcaag gggtcgctgg tattcgtgca gggcaagatt cggaatacca
 2821 agtacgagaa ggacggccag acggtctacg ggaccgactt cattgccgat aaggtggatt
 2881 atctggacac caaggcacca ggcgggtcaa atcaggaata agggcacatt gccccggcgt
 2941 gagtcggggc aatcccgcaa ggagggtgaa tgaatcggac gtttgaccgg aaggcataca
 3001 ggcaagaact gatcgacgcg gggttttccg ccgaggatgc cgaaaccatc gcaagccgca
 3061 ccgtcatgcg tgcgccccgc gaaaccttcc agtccgtcgg ctcgatggtc cagcaagcta
 3121 cggccaagat cgagcgcgac agcgtgcaac tggctccccc tgccctgccc gcgccatcgg
 3181 ccgccgtgga gcgttcgcgt cgtcttgaac aggaggcggc aggtttggcg aagtcgatga
 3241 ccatcgacac gcgaggaact atgacgacca agaagcgaaa aaccgccggc gaggacctgg
 3301 caaaacaggt cagcgaggcc aagcaggccg cgttgctgaa acacacgaag cagcagatca
 3361 aggaaatgca gctttccttg ttcgatattg cgccgtggcc ggacacgatg cgagcgatgc
 3421 caaacgacac ggcccgctct gccctgttca ccacgcgcaa caagaaaatc ccgcgcgagg
 3481 cgctgcaaaa caaggtcatt ttccacgtca acaaggacgt gaagatcacc tacaccggcg
 3541 tcgagctgcg ggccgacgat gacgaactgg tgtggcagca ggtgttggag tacgcgaagc
 3601 gcacccctat cggcgagccg atcaccttca cgttctacga gctttgccag gacctgggct
 3661 ggtcgatcaa tggccggtat tacacgaagg ccgaggaatg cctgtcgcgc ctacaggcga
 3721 cggcgatggg cttcacgtcc gaccgcgttg ggcacctgga atcggtgtcg ctgctgcacc
 3781 gcttccgcgt cctggaccgt ggcaagaaaa cgtcccgttg ccaggtcctg atcgacgagg
 3841 aaatcgtcgt gctgtttgct ggcgaccact acacgaaatt catatgggag aagtaccgca
 3901 agctgtcgcc gacggcccga cggatgttcg actatttcag ctcgcaccgg gagccgtacc
 3961 cgctcaagct ggaaaccttc cgcctcatgt gcggatcgga ttccacccgc gtgaagaagt
 4021 ggcgcgagca ggtcggcgaa gcctgcgaag agttgcgagg cagcggcctg gtggaacacg
 4081 cctgggtcaa tgatgacctg gtgcattgca aacgctaggg ccttgtgggg tcagttccgg
 4141 ctgggggttc agcagccagc gcctgatctg gggaaccctg tggttggcat gcacatacaa
 4201 atggacgaac ggataaacct tttcacgccc ttttaaatat ccgattattc taataaacgc
 4261 tcttttctct taggtttacc cgccaatata tcctgtcaaa cactgatagt ttaaaccact
 4321 tcgtgcagaa gacaattgca gcgtgagacc gtcacagctt gtctgtaagc ggatgccggg
 4381 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
 4441 tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac
 4501 agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt
 4561 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
 4621 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
 4681 acggccagtg aattcgagct cggtacccgg ggatcctcta gagtcgacct gcaggcatgc
 4741 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
 4801 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
 4861 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcggcaa acctgtcgtg
 4921 ccagctgcgg tctcactccg gatccgaatt cggcattgtc ttcacaga