LOCUS       sequence-152875-        6630 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas9C in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6630)
  AUTHORS   Chew WL, Tabebordbar M, Cheng JK, Mali P, Wu EY, Ng AH, Zhu K, 
            Wagers AJ, Church GM
  TITLE     A multifunctional AAV-CRISPR-Cas9 and its host response.
  JOURNAL   Nat Methods. 2016 Sep 5. doi: 10.1038/nmeth.3993.
  PUBMED    27595405
REFERENCE   2  (bases 1 to 6630)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2016 Sep 5. doi: 10.1038/nmeth.3993."
COMMENT     Sequence Label: pAAV-SMVP-Cas9C
FEATURES             Location/Qualifiers
     source          1..6630
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     84..103
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    471..492
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(507..512,513..530,531..537)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    545..561
                     /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)."
     primer_bind     550..572
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     569..585
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     569..585
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   612..741
                     /label=AAV2 ITR
     primer_bind     complement(795..815)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     enhancer        1045..1424
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1425..1628
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1579..1599
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          1720..1852
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      1882..1891
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1886..1895
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2492..2503
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             4021..4041
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4052..4071)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    4081..4162
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4106..4125
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4301..4430
                     /label=AAV2 ITR
     primer_bind     complement(4449..4466)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4449..4465)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4458..4480)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4607..5062
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4694..4713)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4904..4925
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5089..5193
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5194..5262,5263..6054)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVVMVTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5412..5431)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6225..6630,1..183)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
       61 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
      121 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
      181 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctgcgg ttttgctcac
      241 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
      301 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
      361 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
      421 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
      481 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
      541 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaga
      601 tttaattaag gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
      661 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      721 actccatcac taggggttcc ttgtagttaa tgattaaccc gccatgctac ttatctacct
      781 gcggccgcca ttatggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
      841 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accagtcccg gtctcctccc
      901 atgcatgtca atattggcca ttagccatat tattcattgg ttatatagca taaatcaata
      961 ttggctattg gccattgcat acgttgtatc tatatcataa tatgtacatt tatattggct
     1021 catgtccaat atgaccgcca tgttggcatt gattattgac tagttattaa tagtaatcaa
     1081 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     1141 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     1201 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     1261 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtccgccc cctattgacg
     1321 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta cgggactttc
     1381 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     1441 agtacaccaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     1501 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     1561 ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     1621 gcagaggtcg tttagtgaac cgtcagatca ctagtagctt tattgcggta gtttatcaca
     1681 gttaaattgc taacgcagtc agtgctcgac tgatcacagg taagtatcaa ggttacaaga
     1741 caggtttaag gaggccaata gaaactgggc ttgtcgagac agagaagatt cttgcgtttc
     1801 tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac aggggtaccg
     1861 aagccgctag cgctaccggt cgccaccatg gcggcggcgt gcccggaact gcgtcagctg
     1921 gcgcagagcg atgtgtattg ggatccgatt gtgagcattg aaccggatgg cgtggaagaa
     1981 gtgtttgatc tgaccgtgcc gggcccgcat aactttgtgg cgaacgatat tattgcgcat
     2041 aactctggcc agggggacag tcttcacgag cacatcgcta atcttgcagg tagcccagct
     2101 atcaaaaagg gaatactgca gaccgttaag gtcgtggatg aactcgtcaa agtaatggga
     2161 aggcataagc ccgagaatat cgttatcgag atggcccgag agaaccaaac tacccagaag
     2221 ggacagaaga acagtaggga aaggatgaag aggattgaag agggtataaa agaactgggg
     2281 tcccaaatcc ttaaggaaca cccagttgaa aacacccagc ttcagaatga gaagctctac
     2341 ctgtactacc tgcagaacgg cagggacatg tacgtggatc aggaactgga catcaatcgg
     2401 ctctccgact acgacgtgga tcatatcgtg ccccagtctt ttctcaaaga tgattctatt
     2461 gataataaag tgttgacaag atccgataaa aatagaggga agagtgataa cgtcccctca
     2521 gaagaagttg tcaagaaaat gaaaaattat tggcggcagc tgctgaacgc caaactgatc
     2581 acacaacgga agttcgataa tctgactaag gctgaacgag gtggcctgtc tgagttggat
     2641 aaagccggct tcatcaaaag gcagcttgtt gagacacgcc agatcaccaa gcacgtggcc
     2701 caaattctcg attcacgcat gaacaccaag tacgatgaaa atgacaaact gattcgagag
     2761 gtgaaagtta ttactctgaa gtctaagctg gtctcagatt tcagaaagga ctttcagttt
     2821 tataaggtga gagagatcaa caattaccac catgcgcatg atgcctacct gaatgcagtg
     2881 gtaggcactg cacttatcaa aaaatatccc aagcttgaat ctgaatttgt ttacggagac
     2941 tataaagtgt acgatgttag gaaaatgatc gcaaagtctg agcaggaaat aggcaaggcc
     3001 accgctaagt acttctttta cagcaatatt atgaattttt tcaagaccga gattacactg
     3061 gccaatggag agattcggaa gcgaccactt atcgaaacaa acggagaaac aggagaaatc
     3121 gtgtgggaca agggtaggga tttcgcgaca gtccggaagg tcctgtccat gccgcaggtg
     3181 aacatcgtta aaaagaccga agtacagacc ggaggcttct ccaaggaaag tatcctcccg
     3241 aaaaggaaca gcgacaagct gatcgcacgc aaaaaagatt gggaccccaa gaaatacggc
     3301 ggattcgatt ctcctacagt cgcttacagt gtactggttg tggccaaagt ggagaaaggg
     3361 aagtctaaaa aactcaaaag cgtcaaggaa ctgctgggca tcacaatcat ggagcgatca
     3421 agcttcgaaa aaaaccccat cgactttctc gaggcgaaag gatataaaga ggtcaaaaaa
     3481 gacctcatca ttaagcttcc caagtactct ctctttgagc ttgaaaacgg ccggaaacga
     3541 atgctcgcta gtgcgggcga gctgcagaaa ggtaacgagc tggcactgcc ctctaaatac
     3601 gttaatttct tgtatctggc cagccactat gaaaagctca aagggtctcc cgaagataat
     3661 gagcagaagc agctgttcgt ggaacaacac aaacactacc ttgatgagat catcgagcaa
     3721 ataagcgaat tctccaaaag agtgatcctc gccgacgcta acctcgataa ggtgctttct
     3781 gcttacaata agcacaggga taagcccatc agggagcagg cagaaaacat tatccacttg
     3841 tttactctga ccaacttggg cgcgcctgca gccttcaagt acttcgacac caccatagac
     3901 agaaagcggt acacctctac aaaggaggtc ctggacgcca cactgattca tcagtcaatt
     3961 acggggctct atgaaacaag aatcgacctc tctcagctcg gtggagacag cagggctgac
     4021 cccaagaaga agaggaaggt gtctcgagct cgatgagttt ggacaaacca caactagaat
     4081 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     4141 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     4201 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtagcggccg
     4261 caggtagata agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag
     4321 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     4381 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ccttaattaa
     4441 cctaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     4501 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     4561 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc
     4621 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     4681 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     4741 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     4801 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     4861 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     4921 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     4981 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     5041 aaaatattaa cgtttataat ttcaggtggc atctttcggg gaaatgtgcg cggaacccct
     5101 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     5161 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5221 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     5281 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     5341 aatagtggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     5401 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     5461 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     5521 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     5581 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     5641 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     5701 gccataccaa acgacgagcg tgacaccacg atgcctgtag taatggtaac aacgttgcgc
     5761 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     5821 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     5881 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     5941 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     6001 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     6061 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6121 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6181 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6241 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6301 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6361 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6421 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6481 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6541 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6601 tacctacagc gtgagctatg agaaagcgcc
//
