LOCUS       pAI4                    7615 bp ds-DNA     circular SYN 08-FEB-2017
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7615)
  AUTHORS   Morten Norholm Group
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Apr 25, 2017 from SnapGene 3.3.3 to Vector NTI(R) 
            format
            http://www.snapgene.com
COMMENT     VNTDATE|795225600|
COMMENT     VNTDBDATE|796953600|
COMMENT     VNTNAME|pAI4|
COMMENT     VNTAUTHORNAME|Morten Norholm Group|
FEATURES             Location/Qualifiers
     terminator      476..523
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             657..1280
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
     misc_feature    1385..1431
                     /label=Doublicate
     CDS             complement(1442..2257)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
     promoter        complement(2258..2349)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    2258..2266
                     /label=USER
     rep_origin      complement(2367..3002)
                     /direction=LEFT
                     /label=ColA ori
                     /note="Plasmids containing the ColA origin of replication 
                     can be propagated in E. coli cells that contain additional 
                     plasmids with compatible origins."
     misc_RNA        complement(3251..3329)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     repeat_region   3501..3536
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   3567..3602
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   3633..3668
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   3699..3734
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   3765..3800
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     protein_bind    3868..3886
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3893..3911
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             3936..427
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
ORIGIN
        1 tctgccaagc aaatatgtga attttttata tttagctagt cattatgaaa agttgaaggg
       61 tagtccagaa gataacgaac aaaaacaatt gtttgtggag cagcataagc attatttaga
      121 tgagattatt gagcaaatca gtgaattttc taagcgtgtt attttagcag atgccaattt
      181 agataaagtt cttagtgcat ataacaaaca tagagacaaa ccaatacgtg aacaagcaga
      241 aaatattatt catttattta cgttgacgaa tcttggagct cccgctgctt ttaaatattt
      301 tgatacaaca attgatcgta aacgatatac gtctacaaaa gaagttttag atgccactct
      361 tatccatcaa tccatcactg gtctttatga aacacgcatt gatttgagtc agctaggagg
      421 tgactgagat cccatgttac cggtatccta ggctgctgcc accgctgagc aataactagc
      481 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaac ctcaggcatt
      541 tgagaagcac acggtcacac tgcttccggt agtcaataaa ccggtattga cgccaacggg
      601 tcacacgggt acagtgctag cagaaacata tccatgaaat cccgccccga attcatatgt
      661 ctagattaga taaaagtaaa gtgattaaca gcgcattaga gctgcttaat gaggtcggaa
      721 tcgaaggttt aacaacccgt aaactcgccc agaagctagg tgtagagcag cctacattgt
      781 attggcatgt aaaaaataag cgggctttgc tcgacgcctt agccattgag atgttagata
      841 ggcaccatac tcacttttgc cctttagaag gggaaagctg gcaagatttt ttacgtaata
      901 aggctaaaag ttttagatgt gctttactaa gtcatcgcga tggagcaaaa gtacatttag
      961 gtacacggcc tacagaaaaa cagtatgaaa ctctcgaaaa tcaattagcc tttttatgcc
     1021 aacaaggttt ttcactagag aatgcattat atgcactcag cgctgtgggg cattttactt
     1081 taggttgcgt attggaagat caagagcatc aagtcgctaa agaagaaagg gaaacaccta
     1141 ctactgatag tatgccgcca ttattacgac aagctatcga attatttgat caccaaggtg
     1201 cagagccagc cttcttattc ggccttgaat tgatcatatg cggattagaa aaacaactta
     1261 aatgtgaaag tgggtcttaa gcaatagaca taagcggcta tttaacgacc ctgccctgaa
     1321 ccgacgacaa gctgacgacc gggtctccgc aagtggcact tttcggggaa atgtgcgcgg
     1381 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     1441 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     1501 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     1561 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     1621 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     1681 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     1741 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     1801 gcgcctgagc gagacgaaat acgcggtcgc tgttaaaagg acaattacaa acaggaatcg
     1861 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     1921 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     1981 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     2041 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     2101 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     2161 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     2221 gcctagagca agacgtttcc cgttgaatat ggctcatact cttccttttt caatattatt
     2281 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     2341 ataaacaaat aggcatgcta gcgcagaaac gtcctagaag atgccaggag gatacttagc
     2401 agagagacaa taaggccgga gcgaagccgt ttttccatag gctccgcccc cctgacgaac
     2461 atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc gacaggacta taaagatacc
     2521 aggcgtttcc ccctgatggc tccctcttgc gctctcctgt tcccgtcctg cggcgtccgt
     2581 gttgtggtgg aggctttacc caaatcacca cgtcccgttc cgtgtagaca gttcgctcca
     2641 agctgggctg tgtgcaagaa ccccccgttc agcccgactg ctgcgcctta tccggtaact
     2701 atcatcttga gtccaacccg gaaagacacg acaaaacgcc actggcagca gccattggta
     2761 actgagaatt agtggattta gatatcgaga gtcttgaagt ggtggcctaa cagaggctac
     2821 actgaaagga cagtatttgg tatctgcgct ccactaaagc cagttaccag gttaagcagt
     2881 tccccaactg acttaacctt cgatcaaacc gcctccccag gcggtttttt cgtttacaga
     2941 gcaggagatt acgacgatcg taaaaggatc tcaagaagat cctttacgga ttcccgacac
     3001 catcactcta gatttcagtg caatttatct cttcaaatgt agcacctgaa gtcagcccca
     3061 tacgatataa gttgtaattc tcatgttagt catgccccgc gcccaccgga aggagctgac
     3121 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     3181 tacattaatt gcgttgcgca caccgactag cgaaaaaacc ccgccgaagc ggggtttttt
     3241 gcgaaaaaaa gcaccgactc ggtgccactt tttcaagttg ataacggact agccttattt
     3301 taacttgcta tgctgttttg aatggttccg ctagcactgt acctaggact gagctagccg
     3361 tcaaaagcag cgtatatacc atggccacaa ttcagcaaat tgtgaacatc atcacgttca
     3421 tctttccctg gttgccaatg gcccattttc ctgtcagtaa cgagaaggtc gcgaattcag
     3481 gcgcttttta gactggtcgt gttttagagc tatgctgttt tgaatggtcc caaaacatga
     3541 actagcgatt agtcgctatg acttaagttt tagagctatg ctgttttgaa tggtcccaaa
     3601 acaaccacac tagagaacat actggctaaa tagttttaga gctatgctgt tttgaatggt
     3661 cccaaaacgg ttggactcaa gacgatagtt accggatagt tttagagcta tgctgttttg
     3721 aatggtccca aaacggcgaa acccgacagg actataaaga taccgtttta gagctatgct
     3781 gttttgaatg gtcccaaaac aaaaaaaaac cccgcccctg acagggcggg gttttttttt
     3841 taattaaagt ctggttataa cctgaggtcc ctatcagtga tagagattga catccctatc
     3901 agtgatagag atactgagca cagaaggaga tatacatgga taagaaatac tcaataggct
     3961 tagatatcgg cacaaatagc gtcggatggg cggtgatcac tgatgaatat aaggttccgt
     4021 ctaaaaagtt caaggttctg ggaaatacag accgccacag tatcaaaaaa aatcttatag
     4081 gggctctttt atttgacagt ggagagacag cggaagcgac tcgtctcaaa cggacagctc
     4141 gtagaaggta tacacgtcgg aagaatcgta tttgttatct acaggagatt ttttcaaatg
     4201 agatggcgaa agtagatgat agtttctttc atcgacttga agagtctttt ttggtggaag
     4261 aagacaagaa gcatgaacgt catcctattt ttggaaatat agtagatgaa gttgcttatc
     4321 atgagaaata tccaactatc tatcatctgc gaaaaaaatt ggtagattct actgataaag
     4381 cggatttgcg cttaatctat ttggccttag cgcatatgat taagtttcgt ggtcattttt
     4441 tgattgaggg agatttaaat cctgataata gtgatgtgga caaactattt atccagttgg
     4501 tacaaaccta caatcaatta tttgaagaaa accctattaa cgcaagtgga gtagatgcta
     4561 aagcgattct ttctgcacga ttgagtaaat caagacgatt agaaaatctc attgctcagc
     4621 tccccggtga gaagaaaaat ggcttatttg ggaatctcat tgctttgtca ttgggtttga
     4681 cccctaattt taaatcaaat tttgatttgg cagaagatgc taaattacag ctttcaaaag
     4741 atacttacga tgatgattta gataatttat tggcgcaaat tggagatcaa tatgctgatt
     4801 tgtttttggc agctaagaat ttatcagatg ctattttact ttcagatatc ctaagagtaa
     4861 atactgaaat aactaaggct cccctatcag cttcaatgat taaacgctac gatgaacatc
     4921 atcaagactt gactctttta aaagctttag ttcgacaaca acttccagaa aagtataaag
     4981 aaatcttttt tgatcaatca aaaaacggat atgcaggtta tattgatggg ggagctagcc
     5041 aagaagaatt ttataaattt atcaaaccaa ttttagaaaa aatggatggt actgaggaat
     5101 tattggtgaa actaaatcgt gaagatttgc tgcgcaagca acggaccttt gacaacggct
     5161 ctattcccca tcaaattcac ttgggtgagc tgcatgctat tttgagaaga caagaagact
     5221 tttatccatt tttaaaagac aatcgtgaga agattgaaaa aatcttgact tttcgaattc
     5281 cttattatgt tggtccattg gcgcgtggca atagtcgttt tgcatggatg actcggaagt
     5341 ctgaagaaac aattacccca tggaattttg aagaagttgt cgataaaggt gcttcagctc
     5401 aatcatttat tgaacgcatg acaaactttg ataaaaatct tccaaatgaa aaagtactac
     5461 caaaacatag tttgctttat gagtatttta cggtttataa cgaattgaca aaggtcaaat
     5521 atgttactga aggaatgcga aaaccagcat ttctttcagg tgaacagaag aaagccattg
     5581 ttgatttact cttcaaaaca aatcgaaaag taaccgttaa gcaattaaaa gaagattatt
     5641 tcaaaaaaat agaatgtttt gatagtgttg aaatttcagg agttgaagat agatttaatg
     5701 cttcattagg tacctaccat gatttgctaa aaattattaa agataaagat tttttggata
     5761 atgaagaaaa tgaagatatc ttagaggata ttgttttaac attgacctta tttgaagata
     5821 gggagatgat tgaggaaaga cttaaaacat atgctcacct ctttgatgat aaggtgatga
     5881 aacagcttaa acgtcgccgt tatactggtt ggggacgttt gtctcgaaaa ttgattaatg
     5941 gtattaggga taagcaatct ggcaaaacaa tattagattt tttgaaatca gatggttttg
     6001 ccaatcgcaa ttttatgcag ctgatccatg atgatagttt gacatttaaa gaagacattc
     6061 aaaaagcaca agtgtctgga caaggcgata gtttacatga acatattgca aatttagctg
     6121 gtagccctgc tattaaaaaa ggtattttac agactgtaaa agttgttgat gaattggtca
     6181 aagtaatggg gcggcataag ccagaaaata tcgttattga aatggcacgt gaaaatcaga
     6241 caactcaaaa gggccagaaa aattcgcgag agcgtatgaa acgaatcgaa gaaggtatca
     6301 aagaattagg aagtcagatt cttaaagagc atcctgttga aaatactcaa ttgcaaaatg
     6361 aaaagctcta tctctattat ctccaaaatg gaagagacat gtatgtggac caagaattag
     6421 atattaatcg tttaagtgat tatgatgtcg atcacattgt tccacaaagt ttccttaaag
     6481 acgattcaat agacaataag gtcttaacgc gttctgataa aaatcgtggt aaatcggata
     6541 acgttccaag tgaagaagta gtcaaaaaga tgaaaaacta ttggagacaa cttctaaacg
     6601 ccaagttaat cactcaacgt aagtttgata atttaacgaa agctgaacgt ggaggtttga
     6661 gtgaacttga taaagctggt tttatcaaac gccaattggt tgaaactcgc caaatcacta
     6721 agcatgtggc acaaattttg gatagtcgca tgaatactaa atacgatgaa aatgataaac
     6781 ttattcgaga ggttaaagtg attaccttaa aatctaaatt agtttctgac ttccgaaaag
     6841 atttccaatt ctataaagta cgtgagatta acaattacca tcatgcccat gatgcgtatc
     6901 taaatgccgt cgttggaact gctttgatta agaaatatcc aaaacttgaa tcggagtttg
     6961 tctatggtga ttataaagtt tatgatgttc gtaaaatgat tgctaagtct gagcaagaaa
     7021 taggcaaagc aaccgcaaaa tatttctttt actctaatat catgaacttc ttcaaaacag
     7081 aaattacact tgcaaatgga gagattcgca aacgccctct aatcgaaact aatggggaaa
     7141 ctggagaaat tgtctgggat aaagggcgag attttgccac agtgcgcaaa gtattgtcca
     7201 tgccccaagt caatattgtc aagaaaacag aagtacagac aggcggattc tccaaggagt
     7261 caattttacc aaaaagaaat tcggacaagc ttattgctcg taaaaaagac tgggatccaa
     7321 aaaaatatgg tggttttgat agtccaacgg tagcttattc agtcctagtg gttgctaagg
     7381 tggaaaaagg gaaatcgaag aagttaaaat ccgttaaaga gttactaggg atcacaatta
     7441 tggaaagaag ttcctttgaa aaaaatccga ttgacttttt agaagctaaa ggatataagg
     7501 aagttaaaaa agacttaatc attaaactac ctaaatatag tctttttgag ttagaaaacg
     7561 gtcgtaaacg gatgctggct agtgccggag aattacaaaa aggaaatgag ctggc
//
