LOCUS       hPGK-ABE7.10-P2A        9387 bp ds-DNA     circular SYN 16-OCT-2020
DEFINITION  expresses ABE7.10 in mammalian cells.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9387)
  AUTHORS   Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu
            DR
  TITLE     Programmable base editing of A*T to G*C in genomic DNA without DNA 
            cleavage.
  JOURNAL   Nature. 2017 Nov 23;551(7681):464-471. doi: 10.1038/nature24644. 
            Epub 2017 Oct 25.
REFERENCE   2  (bases 1 to 9387)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 16, 2020 from SnapGene Viewer 4.2.6 to Vector NTI(R) 
            format
            http://www.snapgene.com
COMMENT     ORIGDB|GenBank
COMMENT     VNTDATE|867369600|
COMMENT     VNTDBDATE|927590400|
COMMENT     VNTNAME|hPGK-ABE7.10-P2A-GFP|
FEATURES             Location/Qualifiers
     source          1..9387
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
                     /label=/vntifkey=98 /vntifkey=98
                     /note="/vntifkey=98 /vntifkey=98"
                     /note="/vntifkey=98"
     CDS             1098..5198
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain 
                     /vntifkey=4"
                     /note="/vntifkey=4"
     promoter        5199..5255
                     /label=P2A
                     /note="/vntifkey=29 /vntifkey=30"
                     /note="/vntifkey=30"
     promoter        5256..5972
                     /label=EGFP
                     /label=GFP
                     /note="/vntifkey=29 /vntifkey=30"
                     /note="/vntifkey=30"
     CDS             5985..6005
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40\NLS
                     /label=SV40 NLS
                     /note="/vntifkey=4 /vntifkey=4"
                     /note="/vntifkey=4"
     CDS             6014..6031
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="/vntifkey=4 /vntifkey=4"
                     /note="/vntifkey=4"
     primer_bind     6054..6071
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse /vntifkey=28 /vntifkey=28"
                     /note="/vntifkey=28"
     polyA_signal    6060..6284
                     /label=bGH\poly(A)\signal
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal 
                     /vntifkey=25"
                     /note="/vntifkey=25"
     primer_bind     6355..6371
                     /label=M13\rev
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants /vntifkey=28 /vntifkey=28"
                     /note="/vntifkey=28"
     primer_bind     6355..6371
                     /label=M13\Reverse
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev /vntifkey=28 
                     /vntifkey=28"
                     /note="/vntifkey=28"
     primer_bind     6368..6390
                     /label=M13/pUC\Reverse
                     /label=M13/pUC Reverse
                     /note="In lacZ gene /vntifkey=28 /vntifkey=28"
                     /note="/vntifkey=28"
     protein_bind    6379..6395
                     /label=lac\operator
                     /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). 
                     /vntifkey=31"
                     /note="/vntifkey=31"
     promoter        complement(6403..6433)
                     /label=lac\promoter
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon /vntifkey=30 
                     /vntifkey=30"
                     /note="/vntifkey=30"
     protein_bind    6448..6469
                     /label=CAP\binding\site
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP. /vntifkey=31 /vntifkey=31"
                     /note="/vntifkey=31"
     primer_bind     6586..6603
                     /label=L4440
                     /note="L4440 vector, forward primer /vntifkey=28 
                     /vntifkey=28"
                     /note="/vntifkey=28"
     rep_origin      complement(6757..7345)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication /vntifkey=33 /vntifkey=33"
                     /note="/vntifkey=33"
     primer_bind     6837..6856
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer /vntifkey=28 
                     /vntifkey=28"
                     /note="/vntifkey=28"
     CDS             complement(7516..8376)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics /vntifkey=4 /vntifkey=4"
                     /note="/vntifkey=4"
     primer_bind     8139..8158
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer 
                     /vntifkey=28"
                     /note="/vntifkey=28"
     promoter        complement(8377..8481)
                     /gene="bla"
                     /label=AmpR\promoter
                     /label=AmpR promoter
                     /note="/vntifkey=30 /vntifkey=30"
                     /note="/vntifkey=30"
     primer_bind     8560..8579
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable marker
                     /vntifkey=28"
                     /note="/vntifkey=28"
     promoter        8745..9255
                     /label=hPGK
                     /note="/vntifkey=29"
     promoter        join(8995..9088,1..1097)
                     /label=TadA-TadA*(7.10)
                     /note="/vntifkey=29"
                     /note="/vntifkey=30"
     primer_bind     9262..9281
                     /label=T7
                     /note="T7 promoter, forward primer /vntifkey=28 
                     /vntifkey=28"
                     /note="/vntifkey=28"
     promoter        9262..9280
                     /label=T7\promoter
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase 
                     /vntifkey=30"
                     /note="/vntifkey=30"
ORIGIN
        1 cagtactcgt gcataacaat cgcgtaatcg gcgaaggttg gaataggccg atcggacgcc
       61 acgaccccac tgcacatgcg gaaatcatgg cccttcgaca gggagggctt gtgatgcaga
      121 attatcgact tatcgatgcg acgctgtacg tcacgcttga accttgcgta atgtgcgcgg
      181 gagctatgat tcactcccgc attggacgag ttgtattcgg tgcccgcgac gccaagacgg
      241 gtgccgcagg ttcactgatg gacgtgctgc atcacccagg catgaaccac cgggtagaaa
      301 tcacagaagg catattggcg gacgaatgtg cggcgctgtt gtccgacttt tttcgcatgc
      361 ggaggcagga gatcaaggcc cagaaaaaag cacaatcctc tactgactct ggtggttctt
      421 ctggtggttc tagcggcagc gagactcccg ggacctcaga gtccgccaca cccgaaagtt
      481 ctggtggttc ttctggtggt tcttccgaag tcgagttttc ccatgagtac tggatgagac
      541 acgcattgac tctcgcaaag agggctcgag atgaacgcga ggtgcccgtg ggggcagtac
      601 tcgtgctcaa caatcgcgta atcggcgaag gttggaatag ggcaatcgga ctccacgacc
      661 ccactgcaca tgcggaaatc atggcccttc gacagggagg gcttgtgatg cagaattatc
      721 gacttatcga tgcgacgctg tacgtcacgt ttgaaccttg cgtaatgtgc gcgggagcta
      781 tgattcactc ccgcattgga cgagttgtat tcggtgttcg caacgccaag acgggtgccg
      841 caggttcact gatggacgtg ctgcattacc caggcatgaa ccaccgggta gaaatcacag
      901 aaggcatatt ggcggacgaa tgtgcggcgc tgttgtgtta cttttttcgc atgcccaggc
      961 aggtctttaa cgcccagaaa aaagcacaat cctctactga ctctggtggt tcttctggtg
     1021 gttctagcgg cagcgagact cccgggacct cagagtccgc cacacccgaa agttctggtg
     1081 gttcttctgg tggttctgat aaaaagtatt ctattggttt agccatcggc actaattccg
     1141 ttggatgggc tgtcataacc gatgaataca aagtaccttc aaagaaattt aaggtgttgg
     1201 ggaacacaga ccgtcattcg attaaaaaga atcttatcgg tgccctccta ttcgatagtg
     1261 gcgaaacggc agaggcgact cgcctgaaac gaaccgctcg gagaaggtat acacgtcgca
     1321 agaaccgaat atgttactta caagaaattt ttagcaatga gatggccaaa gttgacgatt
     1381 ctttctttca ccgtttggaa gagtccttcc ttgtcgaaga ggacaagaaa catgaacggc
     1441 accccatctt tggaaacata gtagatgagg tggcatatca tgaaaagtac ccaacgattt
     1501 atcacctcag aaaaaagcta gttgactcaa ctgataaagc ggacctgagg ttaatctact
     1561 tggctcttgc ccatatgata aagttccgtg ggcactttct cattgagggt gatctaaatc
     1621 cggacaactc ggatgtcgac aaactgttca tccagttagt acaaacctat aatcagttgt
     1681 ttgaagagaa ccctataaat gcaagtggcg tggatgcgaa ggctattctt agcgcccgcc
     1741 tctctaaatc ccgacggcta gaaaacctga tcgcacaatt acccggagag aagaaaaatg
     1801 ggttgttcgg taaccttata gcgctctcac taggcctgac accaaatttt aagtcgaact
     1861 tcgacttagc tgaagatgcc aaattgcagc ttagtaagga cacgtacgat gacgatctcg
     1921 acaatctact ggcacaaatt ggagatcagt atgcggactt atttttggct gccaaaaacc
     1981 ttagcgatgc aatcctccta tctgacatac tgagagttaa tactgagatt accaaggcgc
     2041 cgttatccgc ttcaatgatc aaaaggtacg atgaacatca ccaagacttg acacttctca
     2101 aggccctagt ccgtcagcaa ctgcctgaga aatataagga aatattcttt gatcagtcga
     2161 aaaacgggta cgcaggttat attgacggcg gagcgagtca agaggaattc tacaagttta
     2221 tcaaacccat attagagaag atggatggga cggaagagtt gcttgtaaaa ctcaatcgcg
     2281 aagatctact gcgaaagcag cggactttcg acaacggtag cattccacat caaatccact
     2341 taggcgaatt gcatgctata cttagaaggc aggaggattt ttatccgttc ctcaaagaca
     2401 atcgtgaaaa gattgagaaa atcctaacct ttcgcatacc ttactatgtg ggacccctgg
     2461 cccgagggaa ctctcggttc gcatggatga caagaaagtc cgaagaaacg attactccat
     2521 ggaattttga ggaagttgtc gataaaggtg cgtcagctca atcgttcatc gagaggatga
     2581 ccaactttga caagaattta ccgaacgaaa aagtattgcc taagcacagt ttactttacg
     2641 agtatttcac agtgtacaat gaactcacga aagttaagta tgtcactgag ggcatgcgta
     2701 aacccgcctt tctaagcgga gaacagaaga aagcaatagt agatctgtta ttcaagacca
     2761 accgcaaagt gacagttaag caattgaaag aggactactt taagaaaatt gaatgcttcg
     2821 attctgtcga gatctccggg gtagaagatc gatttaatgc gtcacttggt acgtatcatg
     2881 acctcctaaa gataattaaa gataaggact tcctggataa cgaagagaat gaagatatct
     2941 tagaagatat agtgttgact cttaccctct ttgaagatcg ggaaatgatt gaggaaagac
     3001 taaaaacata cgctcacctg ttcgacgata aggttatgaa acagttaaag aggcgtcgct
     3061 atacgggctg gggacgattg tcgcggaaac ttatcaacgg gataagagac aagcaaagtg
     3121 gtaaaactat tctcgatttt ctaaagagcg acggcttcgc caataggaac tttatgcagc
     3181 tgatccatga tgactcttta accttcaaag aggatataca aaaggcacag gtttccggac
     3241 aaggggactc attgcacgaa catattgcga atcttgctgg ttcgccagcc atcaaaaagg
     3301 gcatactcca gacagtcaaa gtagtggatg agctagttaa ggtcatggga cgtcacaaac
     3361 cggaaaacat tgtaatcgag atggcacgcg aaaatcaaac gactcagaag gggcaaaaaa
     3421 acagtcgaga gcggatgaag agaatagaag agggtattaa agaactgggc agccagatct
     3481 taaaggagca tcctgtggaa aatacccaat tgcagaacga gaaactttac ctctattacc
     3541 tacaaaatgg aagggacatg tatgttgatc aggaactgga cataaaccgt ttatctgatt
     3601 acgacgtcga tcacattgta ccccaatcct ttttgaagga cgattcaatc gacaataaag
     3661 tgcttacacg ctcggataag aaccgaggga aaagtgacaa tgttccaagc gaggaagtcg
     3721 taaagaaaat gaagaactat tggcggcagc tcctaaatgc gaaactgata acgcaaagaa
     3781 agttcgataa cttaactaaa gctgagaggg gtggcttgtc tgaacttgac aaggccggat
     3841 ttattaaacg tcagctcgtg gaaacccgcc aaatcacaaa gcatgttgca cagatactag
     3901 attcccgaat gaatacgaaa tacgacgaga acgataagct gattcgggaa gtcaaagtaa
     3961 tcactttaaa gtcaaaattg gtgtcggact tcagaaagga ttttcaattc tataaagtta
     4021 gggagataaa taactaccac catgcgcacg acgcttatct taatgccgtc gtagggaccg
     4081 cactcattaa gaaatacccg aagctagaaa gtgagtttgt gtatggtgat tacaaagttt
     4141 atgacgtccg taagatgatc gcgaaaagcg aacaggagat aggcaaggct acagccaaat
     4201 acttctttta ttctaacatt atgaatttct ttaagacgga aatcactctg gcaaacggag
     4261 agatacgcaa acgaccttta attgaaacca atggggagac aggtgaaatc gtatgggata
     4321 agggccggga cttcgcgacg gtgagaaaag ttttgtccat gccccaagtc aacatagtaa
     4381 agaaaactga ggtgcagacc ggagggtttt caaaggaatc gattcttcca aaaaggaata
     4441 gtgataagct catcgctcgt aaaaaggact gggacccgaa aaagtacggt ggcttcgata
     4501 gccctacagt tgcctattct gtcctagtag tggcaaaagt tgagaaggga aaatccaaga
     4561 aactgaagtc agtcaaagaa ttattgggga taacgattat ggagcgctcg tcttttgaaa
     4621 agaaccccat cgacttcctt gaggcgaaag gttacaagga agtaaaaaag gatctcataa
     4681 ttaaactacc aaagtatagt ctgtttgagt tagaaaatgg ccgaaaacgg atgttggcta
     4741 gcgccggaga gcttcaaaag gggaacgaac tcgcactacc gtctaaatac gtgaatttcc
     4801 tgtatttagc gtcccattac gagaagttga aaggttcacc tgaagataac gaacagaagc
     4861 aactttttgt tgagcagcac aaacattatc tcgacgaaat catagagcaa atttcggaat
     4921 tcagtaagag agtcatccta gctgatgcca atctggacaa agtattaagc gcatacaaca
     4981 agcacaggga taaacccata cgtgagcagg cggaaaatat tatccatttg tttactctta
     5041 ccaacctcgg cgctccagcc gcattcaagt attttgacac aacgatagat cgcaaacgat
     5101 acacttctac caaggaggtg ctagacgcga cactgattca ccaatccatc acgggattat
     5161 atgaaactcg gatagatttg tcacagcttg ggggtgacgc tactaacttc agcctgctga
     5221 agcaggctgg agacgtggag gagaaccctg gacctatggt gagcaagggc gaggagctgt
     5281 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     5341 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     5401 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctatggag
     5461 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     5521 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     5581 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     5641 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     5701 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
     5761 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     5821 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga
     5881 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     5941 ggatcactct cggcatggac gagctgtaca agtctggtgg ttctcccaag aagaagagga
     6001 aagtctaacc ggtcatcatc accatcacca ttgagtttaa acccgctgat cagcctcgac
     6061 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     6121 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     6181 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     6241 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     6301 aaccagctgg ggctcgatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     6361 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     6421 cataaagtgt aaagcctagg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     6481 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     6541 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     6601 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     6661 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     6721 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     6781 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     6841 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     6901 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     6961 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     7021 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     7081 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     7141 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     7201 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     7261 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     7321 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     7381 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     7441 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     7501 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     7561 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     7621 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     7681 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     7741 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     7801 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     7861 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     7921 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     7981 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     8041 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     8101 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     8161 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     8221 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     8281 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     8341 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     8401 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     8461 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
     8521 gggagatcga tctcccgatc ccctagggtc gactctcagt acaatctgct ctgatgccgc
     8581 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
     8641 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
     8701 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tataggggtt ggggttgcgc
     8761 cttttccaag gcagccctgg gtttgcgcag ggacgcggct gctctgggcg tggttccggg
     8821 aaacgcagcg gcgccgaccc tgggtctcgc acattcttca cgtccgttcg cagcgtcacc
     8881 cggatcttcg ccgctaccct tgtgggcccc ccggcgacgc ttcctgctcc gcccctaagt
     8941 cgggaaggtt ccttgcggtt cgcggcgtgc cggacgtgac aaacggaagc cgcacgtctc
     9001 actagtaccc tcgcagacgg acagcgccag ggagcaatgg cagcgcgccg accgcgatgg
     9061 gctgtggcca atagcggctg ctcagcaggg cgcgccgaga gcagcggccg ggaaggggcg
     9121 gtgcgggagg cggggtgtgg ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc
     9181 gcattctgca agcctccgga gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc
     9241 gacctctctc cccagggccg ctaatacgac tcactatagg gagagccgcc accatgtccg
     9301 aagtcgagtt ttcccatgag tactggatga gacacgcatt gactctcgca aagagggctt
     9361 gggatgaacg cgaggtgccc gtggggg
//
