LOCUS       Exported                9391 bp ds-DNA     circular SYN 25-MAY-2017
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pET28a-Cas9-His
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9391)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, May 30, 2017 from SnapGene 3.3.4
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9391
                     /organism="pET-28 a (+)"
                     /mol_type="other DNA"
     gene            complement(560..1375)
                     /gene="KanR2"
                     /label=KanR2
     CDS             complement(560..1375)
                     /codon_start=1
                     /label=ORF frame 2
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      1481..2100
                     /label=pBR322_ origin
                     /label=pBR322_origin
     misc_feature    2499..2521
                     /label=pGEX_3_primer
     misc_feature    complement(2515..2706)
                     /label=ROP
     gene            complement(3181..3456)
                     /gene="tet (576 - 851)"
                     /label=tet (576 - 851)
     misc_feature    complement(3515..4606)
                     /label=lacI
     CDS             complement(3515..4474)
                     /codon_start=1
                     /label=ORF frame 2
                     /translation="MAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSR
                     ADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPA
                     LFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKY
                     LTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLR
                     VGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVS
                     LVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ"
     CDS             complement(4347..4847)
                     /codon_start=1
                     /label=ORF frame 1
                     /translation="MSACFGVGMVAGPVAGGLLGAISLHAPFLAAAVLNGLNLLLGCFL
                     MQESHKGERRDPGHHRMAQNLSRYGMIAPGRESIQGGECETSNVIRCRRVCRCLLSDRF
                     PRGEPGQPRFCENAGKSGSGDGGAELHSQPRGTTTGGQTVVADWRCHLQSGPARAVANC
                     RGD"
     gene            complement(4689..4952)
                     /gene="tet (300 - 563)"
                     /label=tet (300 - 563)
     misc_feature    4881..4900
                     /label=pBRrevBam_primer
     promoter        4984..5002
                     /label=T7_ promoter
                     /label=T7_promoter
     misc_feature    5002..5029
                     /label=lacO
     misc_feature    5047..5063
                     /label=T7_transl_en_RBS
     misc_feature    complement(5069..5074)
                     /note=""
     misc_feature    5080..5103
                     /label=SV40 NLS
     misc_feature    5089..5103
                     /label=NLS
     misc_feature    5104..9207
                     /note="spCas9"
     misc_feature    9208..9228
                     /note="NLS"
     terminator      9263..9391
                     /label=T7_ terminator
                     /label=T7_terminator
     misc_feature    complement(9305..9323)
                     /label=T7_Terminal_primer
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt
      481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
      541 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
      601 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
      661 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
      721 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
      781 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
      841 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
      901 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
      961 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     1021 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     1081 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     1141 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     1201 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     1261 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     1321 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     1381 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     1441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     1501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     1561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     1621 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     1681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     1741 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     1801 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     1861 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     1921 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     1981 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2041 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2101 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     2161 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     2221 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     2281 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta
     2341 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     2401 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     2461 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     2521 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     2581 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     2641 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     2701 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     2761 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     2821 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     2881 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     2941 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     3001 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     3061 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     3121 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     3181 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     3241 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     3301 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     3361 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     3421 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     3481 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3541 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3601 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     3661 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     3721 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     3781 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     3841 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     3901 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     3961 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     4021 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     4081 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     4141 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     4201 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     4261 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     4321 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     4381 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     4441 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     4501 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     4561 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     4621 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     4681 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     4741 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     4801 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     4861 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     4921 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
     4981 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
     5041 ttttgtttaa ctttaagaag gagatatacc atggggttta tgcccaagaa gaagaggaag
     5101 gtgatggata agaaatactc aataggctta gatatcggca caaatagcgt cggatgggcg
     5161 gtgatcactg atgaatataa ggttccgtct aaaaagttca aggttctggg aaatacagac
     5221 cgccacagta tcaaaaaaaa tcttataggg gctcttttat ttgacagtgg agagacagcg
     5281 gaagcgactc gtctcaaacg gacagctcgt agaaggtata cacgtcggaa gaatcgtatt
     5341 tgttatctac aggagatttt ttcaaatgag atggcgaaag tagatgatag tttctttcat
     5401 cgacttgaag agtctttttt ggtggaagaa gacaagaagc atgaacgtca tcctattttt
     5461 ggaaatatag tagatgaagt tgcttatcat gagaaatatc caactatcta tcatctgcga
     5521 aaaaaattgg tagattctac tgataaagcg gatttgcgct taatctattt ggccttagcg
     5581 catatgatta agtttcgtgg tcattttttg attgagggag atttaaatcc tgataatagt
     5641 gatgtggaca aactatttat ccagttggta caaacctaca atcaattatt tgaagaaaac
     5701 cctattaacg caagtggagt agatgctaaa gcgattcttt ctgcacgatt gagtaaatca
     5761 agacgattag aaaatctcat tgctcagctc cccggtgaga agaaaaatgg cttatttggg
     5821 aatctcattg ctttgtcatt gggtttgacc cctaatttta aatcaaattt tgatttggca
     5881 gaagatgcta aattacagct ttcaaaagat acttacgatg atgatttaga taatttattg
     5941 gcgcaaattg gagatcaata tgctgatttg tttttggcag ctaagaattt atcagatgct
     6001 attttacttt cagatatcct aagagtaaat actgaaataa ctaaggctcc cctatcagct
     6061 tcaatgatta aacgctacga tgaacatcat caagacttga ctcttttaaa agctttagtt
     6121 cgacaacaac ttccagaaaa gtataaagaa atcttttttg atcaatcaaa aaacggatat
     6181 gcaggttata ttgatggggg agctagccaa gaagaatttt ataaatttat caaaccaatt
     6241 ttagaaaaaa tggatggtac tgaggaatta ttggtgaaac taaatcgtga agatttgctg
     6301 cgcaagcaac ggacctttga caacggctct attccccatc aaattcactt gggtgagctg
     6361 catgctattt tgagaagaca agaagacttt tatccatttt taaaagacaa tcgtgagaag
     6421 attgaaaaaa tcttgacttt tcgaattcct tattatgttg gtccattggc gcgtggcaat
     6481 agtcgttttg catggatgac tcggaagtct gaagaaacaa ttaccccatg gaattttgaa
     6541 gaagttgtcg ataaaggtgc ttcagctcaa tcatttattg aacgcatgac aaactttgat
     6601 aaaaatcttc caaatgaaaa agtactacca aaacatagtt tgctttatga gtattttacg
     6661 gtttataacg aattgacaaa ggtcaaatat gttactgaag gaatgcgaaa accagcattt
     6721 ctttcaggtg aacagaagaa agccattgtt gatttactct tcaaaacaaa tcgaaaagta
     6781 accgttaagc aattaaaaga agattatttc aaaaaaatag aatgttttga tagtgttgaa
     6841 atttcaggag ttgaagatag atttaatgct tcattaggta cctaccatga tttgctaaaa
     6901 attattaaag ataaagattt tttggataat gaagaaaatg aagatatctt agaggatatt
     6961 gttttaacat tgaccttatt tgaagatagg gagatgattg aggaaagact taaaacatat
     7021 gctcacctct ttgatgataa ggtgatgaaa cagcttaaac gtcgccgtta tactggttgg
     7081 ggacgtttgt ctcgaaaatt gattaatggt attagggata agcaatctgg caaaacaata
     7141 ttagattttt tgaaatcaga tggttttgcc aatcgcaatt ttatgcagct gatccatgat
     7201 gatagtttga catttaaaga agacattcaa aaagcacaag tgtctggaca aggcgatagt
     7261 ttacatgaac atattgcaaa tttagctggt agccctgcta ttaaaaaagg tattttacag
     7321 actgtaaaag ttgttgatga attggtcaaa gtaatggggc ggcataagcc agaaaatatc
     7381 gttattgaaa tggcacgtga aaatcagaca actcaaaagg gccagaaaaa ttcgcgagag
     7441 cgtatgaaac gaatcgaaga aggtatcaaa gaattaggaa gtcagattct taaagagcat
     7501 cctgttgaaa atactcaatt gcaaaatgaa aagctctatc tctattatct ccaaaatgga
     7561 agagacatgt atgtggacca agaattagat attaatcgtt taagtgatta tgatgtcgat
     7621 cacattgttc cacaaagttt ccttaaagac gattcaatag acaataaggt cttaacgcgt
     7681 tctgataaaa atcgtggtaa atcggataac gttccaagtg aagaagtagt caaaaagatg
     7741 aaaaactatt ggagacaact tctaaacgcc aagttaatca ctcaacgtaa gtttgataat
     7801 ttaacgaaag ctgaacgtgg aggtttgagt gaacttgata aagctggttt tatcaaacgc
     7861 caattggttg aaactcgcca aatcactaag catgtggcac aaattttgga tagtcgcatg
     7921 aatactaaat acgatgaaaa tgataaactt attcgagagg ttaaagtgat taccttaaaa
     7981 tctaaattag tttctgactt ccgaaaagat ttccaattct ataaagtacg tgagattaac
     8041 aattaccatc atgcccatga tgcgtatcta aatgccgtcg ttggaactgc tttgattaag
     8101 aaatatccaa aacttgaatc ggagtttgtc tatggtgatt ataaagttta tgatgttcgt
     8161 aaaatgattg ctaagtctga gcaagaaata ggcaaagcaa ccgcaaaata tttcttttac
     8221 tctaatatca tgaacttctt caaaacagaa attacacttg caaatggaga gattcgcaaa
     8281 cgccctctaa tcgaaactaa tggggaaact ggagaaattg tctgggataa agggcgagat
     8341 tttgccacag tgcgcaaagt attgtccatg ccccaagtca atattgtcaa gaaaacagaa
     8401 gtacagacag gcggattctc caaggagtca attttaccaa aaagaaattc ggacaagctt
     8461 attgctcgta aaaaagactg ggatccaaaa aaatatggtg gttttgatag tccaacggta
     8521 gcttattcag tcctagtggt tgctaaggtg gaaaaaggga aatcgaagaa gttaaaatcc
     8581 gttaaagagt tactagggat cacaattatg gaaagaagtt cctttgaaaa aaatccgatt
     8641 gactttttag aagctaaagg atataaggaa gttaaaaaag acttaatcat taaactacct
     8701 aaatatagtc tttttgagtt agaaaacggt cgtaaacgga tgctggctag tgccggagaa
     8761 ttacaaaaag gaaatgagct ggctctgcca agcaaatatg tgaatttttt atatttagct
     8821 agtcattatg aaaagttgaa gggtagtcca gaagataacg aacaaaaaca attgtttgtg
     8881 gagcagcata agcattattt agatgagatt attgagcaaa tcagtgaatt ttctaagcgt
     8941 gttattttag cagatgccaa tttagataaa gttcttagtg catataacaa acatagagac
     9001 aaaccaatac gtgaacaagc agaaaatatt attcatttat ttacgttgac gaatcttgga
     9061 gctcccgctg cttttaaata ttttgataca acaattgatc gtaaacgata tacgtctaca
     9121 aaagaagttt tagatgccac tcttatccat caatccatca ctggtcttta tgaaacacgc
     9181 attgatttga gtcagctagg aggtgacccc aagaagaaga ggaaggtgct cgagcaccac
     9241 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     9301 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     9361 tttttgctga aaggaggaac tatatccgga t
//
