LOCUS       sequence-155820-        6849 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas9C in mammalian cells; derived from pZac2.1 with CASI 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6849)
  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 6849)
  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-CASI-Cas9C
FEATURES             Location/Qualifiers
     source          1..6849
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     enhancer        206..509
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        511..788
                     /label=chicken beta-actin promoter
     regulatory      1262..1271
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1266..1275
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1872..1883
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3401..3421
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3448..3503
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3502..3521)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   3909..4038
                     /label=AAV2 ITR
     primer_bind     complement(4057..4074)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4057..4073)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4066..4088)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4215..4670
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4302..4321)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4512..4533
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4697..4801
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4802..4870,4871..5662)
                     /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(5020..5039)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5833..6421
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6322..6341
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    6709..6730
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6745..6750,6751..6768,6769..6775)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6783..6799
                     /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     6788..6810
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6807..6823
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6807..6823
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct
      181 aggaagatcg gaattcggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      241 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      301 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      361 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      421 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      481 tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc
      541 tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt
      601 gtgcagcgat gggggcgggg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg
      661 aggggcgggg cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc
      721 gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc
      781 ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc
      841 gccgcccgcc ccggctctga ctgaccgcgt tactaaaaca ggtaagtccg gcctccgcgc
      901 cgggttttgg cgcctcccgc gggcgccccc ctcctcacgg cgagcgctgc cacgtcagac
      961 gaagggcgca gcgagcgtcc tgatccttcc gcccggacgc tcaggacagc ggcccgctgc
     1021 tcataagact cggccttaga accccagtat cagcagaagg acattttagg acgggacttg
     1081 ggtgactcta gggcactggt tttctttcca gagagcggaa caggcgagga aaagtagtcc
     1141 cttctcggcg attctgcgga gggatctccg tggggcggtg aacgccgatg atgcctctac
     1201 taaccatgtt catgttttct ttttttttct acaggtcctg ggtgacgaac aggctagcgc
     1261 cgccaccatg gcggcggcgt gcccggaact gcgtcagctg gcgcagagcg atgtgtattg
     1321 ggatccgatt gtgagcattg aaccggatgg cgtggaagaa gtgtttgatc tgaccgtgcc
     1381 gggcccgcat aactttgtgg cgaacgatat tattgcgcat aactctggcc agggggacag
     1441 tcttcacgag cacatcgcta atcttgcagg tagcccagct atcaaaaagg gaatactgca
     1501 gaccgttaag gtcgtggatg aactcgtcaa agtaatggga aggcataagc ccgagaatat
     1561 cgttatcgag atggcccgag agaaccaaac tacccagaag ggacagaaga acagtaggga
     1621 aaggatgaag aggattgaag agggtataaa agaactgggg tcccaaatcc ttaaggaaca
     1681 cccagttgaa aacacccagc ttcagaatga gaagctctac ctgtactacc tgcagaacgg
     1741 cagggacatg tacgtggatc aggaactgga catcaatcgg ctctccgact acgacgtgga
     1801 tcatatcgtg ccccagtctt ttctcaaaga tgattctatt gataataaag tgttgacaag
     1861 atccgataaa aatagaggga agagtgataa cgtcccctca gaagaagttg tcaagaaaat
     1921 gaaaaattat tggcggcagc tgctgaacgc caaactgatc acacaacgga agttcgataa
     1981 tctgactaag gctgaacgag gtggcctgtc tgagttggat aaagccggct tcatcaaaag
     2041 gcagcttgtt gagacacgcc agatcaccaa gcacgtggcc caaattctcg attcacgcat
     2101 gaacaccaag tacgatgaaa atgacaaact gattcgagag gtgaaagtta ttactctgaa
     2161 gtctaagctg gtctcagatt tcagaaagga ctttcagttt tataaggtga gagagatcaa
     2221 caattaccac catgcgcatg atgcctacct gaatgcagtg gtaggcactg cacttatcaa
     2281 aaaatatccc aagcttgaat ctgaatttgt ttacggagac tataaagtgt acgatgttag
     2341 gaaaatgatc gcaaagtctg agcaggaaat aggcaaggcc accgctaagt acttctttta
     2401 cagcaatatt atgaattttt tcaagaccga gattacactg gccaatggag agattcggaa
     2461 gcgaccactt atcgaaacaa acggagaaac aggagaaatc gtgtgggaca agggtaggga
     2521 tttcgcgaca gtccggaagg tcctgtccat gccgcaggtg aacatcgtta aaaagaccga
     2581 agtacagacc ggaggcttct ccaaggaaag tatcctcccg aaaaggaaca gcgacaagct
     2641 gatcgcacgc aaaaaagatt gggaccccaa gaaatacggc ggattcgatt ctcctacagt
     2701 cgcttacagt gtactggttg tggccaaagt ggagaaaggg aagtctaaaa aactcaaaag
     2761 cgtcaaggaa ctgctgggca tcacaatcat ggagcgatca agcttcgaaa aaaaccccat
     2821 cgactttctc gaggcgaaag gatataaaga ggtcaaaaaa gacctcatca ttaagcttcc
     2881 caagtactct ctctttgagc ttgaaaacgg ccggaaacga atgctcgcta gtgcgggcga
     2941 gctgcagaaa ggtaacgagc tggcactgcc ctctaaatac gttaatttct tgtatctggc
     3001 cagccactat gaaaagctca aagggtctcc cgaagataat gagcagaagc agctgttcgt
     3061 ggaacaacac aaacactacc ttgatgagat catcgagcaa ataagcgaat tctccaaaag
     3121 agtgatcctc gccgacgcta acctcgataa ggtgctttct gcttacaata agcacaggga
     3181 taagcccatc agggagcagg cagaaaacat tatccacttg tttactctga ccaacttggg
     3241 cgcgcctgca gccttcaagt acttcgacac caccatagac agaaagcggt acacctctac
     3301 aaaggaggtc ctggacgcca cactgattca tcagtcaatt acggggctct atgaaacaag
     3361 aatcgacctc tctcagctcg gtggagacag cagggctgac cccaagaaga agaggaaggt
     3421 gtctcgagct cgatgaaccg gttggctaat aaaggaaatt tattttcatt gcaatagtgt
     3481 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac
     3541 atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga
     3601 acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct
     3661 tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat
     3721 ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc
     3781 tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatcg gatccgaatt
     3841 cccgataagg atcttcctag agcatggcta cgtagataag tagcatggcg ggttaatcat
     3901 taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     3961 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     4021 gagcgagcga gcgcgcagcc ttaattaacc taattcactg gccgtcgttt tacaacgtcg
     4081 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     4141 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     4201 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4261 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     4321 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4381 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     4441 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     4501 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     4561 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     4621 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttataattt caggtggcat
     4681 ctttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4741 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4801 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     4861 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     4921 acgagtgggt tacatcgaac tggatctcaa tagtggtaag atccttgaga gttttcgccc
     4981 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5041 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5101 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5161 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5221 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5281 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     5341 gcctgtagta atggtaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     5401 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     5461 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     5521 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     5581 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5641 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     5701 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     5761 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     5821 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     5881 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     5941 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     6001 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6061 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6121 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6181 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6241 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6301 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     6361 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     6421 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctgcggtt ttgctcacat
     6481 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     6541 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     6601 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     6661 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     6721 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     6781 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccagatt
     6841 taattaagg
//
