LOCUS       Exported               10177 bp ds-DNA     circular SYN 01-MAR-2017
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pnCas9-PBE
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10177)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, May 30, 2017 from SnapGene 3.3.4
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..10177
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    13..1999
                     /label=ubi promoter
     CDS             2021..2041
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    2042..2722
                     /label=rAPOBEC1
     misc_feature    2723..2770
                     /label=XTEN
     misc_feature    2777..6925
                     /label=nCas9(D10A)
     CDS             6878..6925
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     misc_feature    6947..7207
                     /label=UGI
     CDS             7208..7228
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    7258..7981
                     /label=CaMV term
     polyA_signal    7302..7478
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     primer_bind     complement(8021..8037)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    8045..8061
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(8069..8099)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8114..8135
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(8423..9011)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(9182..10042)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(10043..10147)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gagctcggta cctgacccgg tcgtgcccct ctctagagat aatgagcatt gcatgtctaa
       61 gttataaaaa attaccacat attttttttg tcacacttgt ttgaagtgca gtttatctat
      121 ctttatacat atatttaaac tttactctac gaataatata atctatagta ctacaataat
      181 atcagtgttt tagagaatca tataaatgaa cagttagaca tggtctaaag gacaattgag
      241 tattttgaca acaggactct acagttttat ctttttagtg tgcatgtgtt ctcctttttt
      301 tttgcaaata gcttcaccta tataatactt catccatttt attagtacat ccatttaggg
      361 tttagggtta atggttttta tagactaatt tttttagtac atctatttta ttctatttta
      421 gcctctaaat taagaaaact aaaactctat tttagttttt ttatttaata atttagatat
      481 aaaatagaat aaaataaagt gactaaaaat taaacaaata ccctttaaga aattaaaaaa
      541 actaaggaaa catttttctt gtttcgagta gataatgcca gcctgttaaa cgccgtcgac
      601 gagtctaacg gacaccaacc agcgaaccag cagcgtcgcg tcgggccaag cgaagcagac
      661 ggcacggcat ctctgtcgct gcctctggac ccctctcgat cgagagttcc gctccaccgt
      721 tggacttgct ccgctgtcgg catccagaaa ttgcgtggcg gagcggcaga cgtgagccgg
      781 cacggcaggc ggcctcctcc tcctctcacg gcaccggcag ctacggggga ttcctttccc
      841 accgctcctt cgctttccct tcctcgcccg ccgtaataaa tagacacccc ctccacaccc
      901 tctttcccca acctcgtgtt gttcggagcg cacacacaca caaccagatc tcccccaaat
      961 ccacccgtcg gcacctccgc ttcaaggtac gccgctcgtc ctcccccccc ccccctctct
     1021 accttctcta gatcggcgtt ccggtccatg gttagggccc ggtagttcta cttctgttca
     1081 tgtttgtgtt agatccgtgt ttgtgttaga tccgtgctgc tagcgttcgt acacggatgc
     1141 gacctgtacg tcagacacgt tctgattgct aacttgccag tgtttctctt tggggaatcc
     1201 tgggatggct ctagccgttc cgcagacggg atcgatttca tgattttttt tgtttcgttg
     1261 catagggttt ggtttgccct tttcctttat ttcaatatat gccgtgcact tgtttgtcgg
     1321 gtcatctttt catgcttttt tttgtcttgg ttgtgatgat gtggtctggt tgggcggtcg
     1381 ttctagatcg gagtagaatt aattctgttt caaactacct ggtggattta ttaattttgg
     1441 atctgtatgt gtgtgccata catattcata gttacgaatt gaagatgatg gatggaaata
     1501 tcgatctagg ataggtatac atgttgatgc gggttttact gatgcatata cagagatgct
     1561 ttttgttcgc ttggttgtga tgatgtggtg tggttgggcg gtcgttcatt cgttctagat
     1621 cggagtagaa tactgtttca aactacctgg tgtatttatt aattttggaa ctgtatgtgt
     1681 gtgtcataca tcttcatagt tacgagttta agatggatgg aaatatcgat ctaggatagg
     1741 tatacatgtt gatgtgggtt ttactgatgc atatacatga tggcatatgc agcatctatt
     1801 catatgctct aaccttgagt acctatctat tataataaac aagtatgttt tataattatt
     1861 ttgatcttga tatacttgga tgatggcata tgcagcagct atatgtggat ttttttagcc
     1921 ctgccttcat acgctattta tttgcttggt actgtttctt ttgtcgatgc tcaccctgtt
     1981 gtttggtgtt acttctgcaa agcttgtcga cggatccatg ccaaagaaga agaggaaggt
     2041 ttcatcggag accggccctg ttgctgttga ccccaccctg cggcggagaa tcgagccaca
     2101 cgagttcgag gtgttcttcg acccaaggga gctccgcaag gagacgtgcc tcctgtacga
     2161 gatcaactgg ggcggcaggc actccatctg gaggcacacc agccaaaaca ccaacaagca
     2221 cgtggaggtc aacttcatcg agaagttcac caccgagagg tacttctgcc caaacacccg
     2281 ctgctccatc acctggttcc tgtcctggag cccatgcggc gagtgctcca gggccatcac
     2341 cgagttcctc agccgctacc cacacgtcac cctgttcatc tacatcgcca ggctctacca
     2401 ccacgccgac ccaaggaaca ggcagggcct ccgcgacctg atctccagcg gcgtgaccat
     2461 ccaaatcatg accgagcagg agtccggcta ctgctggagg aacttcgtca actactcccc
     2521 aagcaacgag gcccactggc caaggtaccc acacctctgg gtgcgcctct acgtgctcga
     2581 gctgtactgc atcatcctcg gcctgccacc atgcctcaac atcctgaggc gcaagcaacc
     2641 acagctgacc ttcttcacca tcgccctcca aagctgccac taccagaggc tcccaccaca
     2701 catcctgtgg gctaccggcc tcaagtccgg cagcgagacg ccaggcacct ccgagagcgc
     2761 tacgcctgaa cttaaggaca agaagtactc gatcggcctc gccatcggga cgaactcagt
     2821 tggctgggcc gtgatcaccg acgagtacaa ggtgccctct aagaagttca aggtcctggg
     2881 gaacaccgac cgccattcca tcaagaagaa cctcatcggc gctctcctgt tcgacagcgg
     2941 ggagaccgct gaggctacga ggctcaagag aaccgctagg cgccggtaca cgagaaggaa
     3001 gaacaggatc tgctacctcc aagagatttt ctccaacgag atggccaagg ttgacgattc
     3061 attcttccac cgcctggagg agtctttcct cgtggaggag gataagaagc acgagcggca
     3121 tcccatcttc ggcaacatcg tggacgaggt tgcctaccac gagaagtacc ctacgatcta
     3181 ccatctgcgg aagaagctcg tggactccac cgataaggcg gacctcagac tgatctacct
     3241 cgctctggcc cacatgatca agttccgcgg ccatttcctg atcgaggggg atctcaaccc
     3301 agacaacagc gatgttgaca agctgttcat ccaactcgtg cagacctaca accaactctt
     3361 cgaggagaac ccgatcaacg cctctggcgt ggacgcgaag gctatcctgt ccgcgaggct
     3421 ctcgaagtcc aggaggctgg agaacctgat cgctcagctc ccaggcgaga agaagaacgg
     3481 cctgttcggg aacctcatcg ctctcagcct ggggctcacc ccgaacttca agtcgaactt
     3541 cgatctcgct gaggacgcca agctgcaact ctccaaggac acctacgacg atgacctcga
     3601 taacctcctg gcccagatcg gcgatcaata cgcggacctg ttcctcgctg ccaagaacct
     3661 gtcggacgcc atcctcctgt cagatatcct ccgcgtgaac accgagatca cgaaggctcc
     3721 actctctgcc tccatgatca agcgctacga cgagcaccat caggatctga ccctcctgaa
     3781 ggcgctggtc cgccaacagc tcccggagaa gtacaaggag attttcttcg atcagtcgaa
     3841 gaacggctac gctgggtaca tcgacggcgg ggcctcacaa gaggagttct acaagttcat
     3901 caagccaatc ctggagaaga tggacggcac ggaggagctc ctggtgaagc tcaacaggga
     3961 ggacctcctg cggaagcaga gaaccttcga taacggcagc atcccccacc aaatccatct
     4021 cggggagctg cacgccatcc tgagaaggca agaggacttc taccctttcc tcaaggataa
     4081 ccgggagaag atcgagaaga tcctgacctt cagaatccca tactacgtcg gccctctcgc
     4141 gcgggggaac tcaagattcg cttggatgac ccgcaagtct gaggagacca tcacgccgtg
     4201 gaacttcgag gaggtggtgg acaagggcgc tagcgctcag tcgttcatcg agaggatgac
     4261 caacttcgac aagaacctgc ccaacgagaa ggtgctccct aagcactcgc tcctgtacga
     4321 gtacttcacc gtctacaacg agctcacgaa ggtgaagtac gtcaccgagg gcatgcgcaa
     4381 gccagcgttc ctgtccgggg agcagaagaa ggctatcgtg gacctcctgt tcaagaccaa
     4441 ccggaaggtc acggttaagc aactcaagga ggactacttc aagaagatcg agtgcttcga
     4501 ttcggtcgag atcagcggcg ttgaggaccg cttcaacgcc agcctcggga cctaccacga
     4561 tctcctgaag atcatcaagg ataaggactt cctggacaac gaggagaacg aggatatcct
     4621 ggaggacatc gtgctgaccc tcacgctgtt cgaggacagg gagatgatcg aggagcgcct
     4681 gaagacgtac gcccatctct tcgatgacaa ggtcatgaag caactcaagc gccggagata
     4741 caccggctgg gggaggctgt cccgcaagct catcaacggc atccgggaca agcagtccgg
     4801 gaagaccatc ctcgacttcc tcaagagcga tggcttcgcc aacaggaact tcatgcaact
     4861 gatccacgat gacagcctca ccttcaagga ggatatccaa aaggctcaag tgagcggcca
     4921 gggggactcg ctgcacgagc atatcgcgaa cctcgctggc tcccccgcga tcaagaaggg
     4981 catcctccag accgtgaagg ttgtggacga gctcgtgaag gtcatgggcc ggcacaagcc
     5041 tgagaacatc gtcatcgaga tggccagaga gaaccaaacc acgcagaagg ggcaaaagaa
     5101 ctctagggag cgcatgaagc gcatcgagga gggcatcaag gagctggggt cccaaatcct
     5161 caaggagcac ccagtggaga acacccaact gcagaacgag aagctctacc tgtactacct
     5221 ccagaacggc agggatatgt acgtggacca agagctggat atcaaccgcc tcagcgatta
     5281 cgacgtcgat catatcgttc cccagtcttt cctgaaggat gactccatcg acaacaaggt
     5341 cctcaccagg tcggacaaga accgcggcaa gtcagataac gttccatctg aggaggtcgt
     5401 taagaagatg aagaactact ggaggcagct cctgaacgcc aagctgatca cgcaaaggaa
     5461 gttcgacaac ctcaccaagg ctgagagagg cgggctctca gagctggaca aggccggctt
     5521 catcaagcgg cagctggtcg agaccagaca aatcacgaag cacgttgcgc aaatcctcga
     5581 ctctcggatg aacacgaagt acgatgagaa cgacaagctg atcagggagg ttaaggtgat
     5641 caccctgaag tctaagctcg tctccgactt caggaaggat ttccagttct acaaggttcg
     5701 cgagatcaac aactaccacc atgcccatga cgcttacctc aacgctgtgg tcggcaccgc
     5761 tctgatcaag aagtacccaa agctggagtc cgagttcgtg tacggggact acaaggttta
     5821 cgatgtgcgc aagatgatcg ccaagtcgga gcaagagatc ggcaaggcta ccgccaagta
     5881 cttcttctac tcaaacatca tgaacttctt caagaccgag atcacgctgg ccaacggcga
     5941 gatccggaag agaccgctca tcgagaccaa cggcgagacg ggggagatcg tgtgggacaa
     6001 gggcagggat ttcgcgaccg tccgcaaggt tctctccatg ccccaggtga acatcgtcaa
     6061 gaagaccgag gtccaaacgg gcgggttctc aaaggagtct atcctgccta agcggaacag
     6121 cgacaagctc atcgccagaa agaaggactg ggacccaaag aagtacggcg ggttcgacag
     6181 ccctaccgtg gcctactcgg tcctggttgt ggcgaaggtt gagaagggca agtccaagaa
     6241 gctcaagagc gtgaaggagc tcctggggat caccatcatg gagaggtcca gcttcgagaa
     6301 gaacccaatc gacttcctgg aggccaaggg ctacaaggag gtgaagaagg acctgatcat
     6361 caagctcccg aagtactctc tcttcgagct ggagaacggc aggaagagaa tgctggcttc
     6421 cgctggcgag ctccagaagg ggaacgagct cgcgctgcca agcaagtacg tgaacttcct
     6481 ctacctggct tcccactacg agaagctcaa gggcagcccg gaggacaacg agcaaaagca
     6541 gctgttcgtc gagcagcaca agcattacct cgacgagatc atcgagcaaa tctccgagtt
     6601 cagcaagcgc gtgatcctcg ccgacgcgaa cctggataag gtcctctccg cctacaacaa
     6661 gcaccgggac aagcccatca gagagcaagc ggagaacatc atccatctct tcaccctgac
     6721 gaacctcggc gctcctgctg ctttcaagta cttcgacacc acgatcgatc ggaagagata
     6781 cacctccacg aaggaggtcc tggacgcgac cctcatccac cagtcgatca ccggcctgta
     6841 cgagacgagg atcgacctct cacaactcgg cggggataag agacccgcag caaccaagaa
     6901 ggcagggcaa gcaaagaaga agaagacgcg tgactccggc ggcagcacca acctgtccga
     6961 catcatcgag aaggagacgg gcaagcaact cgtgatccag gagagcatcc tcatgctgcc
     7021 agaggaggtg gaggaggtca tcggcaacaa gccagagtcc gacatcctgg tgcacaccgc
     7081 ctacgacgag tccaccgacg agaacgtcat gctcctgacc agcgacgccc cagagtacaa
     7141 gccatgggcc ctcgtcatcc aggacagcaa cggggagaac aagatcaaga tgctgtcggg
     7201 ggggagccca aagaagaagc ggaaggtgta gtgtacaagt aacccgggaa ttcggtacgc
     7261 tgaaatcacc agtctctctc tacaaatcta tctctctcta ttttctccat aaataatgtg
     7321 tgagtagttt cccgataagg gaaattaggg ttcttatagg gtttcgctca tgtgttgagc
     7381 atataagaaa cccttagtat gtatttgtat ttgtaaaata cttctatcaa taaaatttct
     7441 aattcctaaa accaaaatcc agtactaaaa tccagatctc ctaaagtccc tatagatctt
     7501 tgtcgtgaat ataaaccaga cacgagacga ctaaacctgg agcccagacg ccgttcgaag
     7561 ctagaagtac cgcttaggca ggaggccgtt agggaaaaga tgctaaggca gggttggtta
     7621 cgttgactcc cccgtaggtt tggtttaaat atgatgaagt ggacggaagg aaggaggaag
     7681 acaaggaagg ataaggttgc aggccctgtg caaggtaaga agatggaaat ttgatagagg
     7741 tacgctacta tacttatact atacgctaag ggaatgcttg tatttatacc ctataccccc
     7801 taataacccc ttatcaattt aagaaataat ccgcataagc ccccgcttaa aaattggtat
     7861 cagagccatg aataggtcta tgaccaaaac tcaagaggat aaaacctcac caaaatacga
     7921 aagagttctt aactctaaag ataaaagatc tttcaagatc aaaactagtt ccctcacacc
     7981 ggagcatgcg atatcctcga gagatctagg cgtaatcatg gtcatagctg tttcctgtgt
     8041 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     8101 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     8161 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     8221 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     8281 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     8341 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     8401 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     8461 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     8521 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     8581 ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt aggtatctca
     8641 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     8701 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     8761 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     8821 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     8881 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     8941 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     9001 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     9061 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     9121 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     9181 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     9241 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     9301 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     9361 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     9421 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     9481 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     9541 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     9601 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     9661 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     9721 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     9781 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     9841 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     9901 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     9961 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
    10021 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
    10081 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
    10141 ttccgcgcac atttccccga aaagtgccac ctgacgt
//
