LOCUS       Exported                7593 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses Cas9C in mammalian cells; co-expresses turboGFP..
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-CASI-Cas9C-P2A-turboGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7593)
  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 7593)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7593
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     regulatory      1266..1275
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     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"
     CDS             3440..3496
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3497..4162
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="MPAMKIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGAL
                     TFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYR
                     YEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDG
                     GYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA
                     "
     polyA_signal    4192..4247
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4246..4265)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   4653..4782
                     /label=AAV2 ITR
     primer_bind     complement(4801..4818)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4801..4817)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4810..4832)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4959..5414
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5046..5065)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5256..5277
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5441..5545
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5546..6406
                     /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(5764..5783)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6577..7165
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7066..7085
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7319..7336
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7453..7474
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7489..7519
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7527..7543
                     /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     7532..7554
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7551..7567
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7551..7567
                     /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 ggatccggag ccacgaactt ctctctgtta aagcaagcag gggacgtgga
     3481 agaaaacccc ggtcctatgc ccgccatgaa gatcgagtgc cgcatcaccg gcaccctgaa
     3541 cggcgtggag ttcgagctgg tgggcggcgg agagggcacc cccgagcagg gccgcatgac
     3601 caacaagatg aagagcacca aaggcgccct gaccttcagc ccctacctgc tgagccacgt
     3661 gatgggctac ggcttctacc acttcggcac ctaccccagc ggctacgaga accccttcct
     3721 gcacgccatc aacaacggcg gctacaccaa cacccgcatc gagaagtacg aggacggcgg
     3781 cgtgctgcac gtgagcttca gctaccgcta cgaggccggc cgcgtgatcg gcgacttcaa
     3841 ggtggtgggc accggcttcc ccgaggacag cgtgatcttc accgacaaga tcatccgcag
     3901 caacgccacc gtggagcacc tgcaccccat gggcgataac gtgctggtgg gcagcttcgc
     3961 ccgcaccttc agcctgcgcg acggcggcta ctacagcttc gtggtggaca gccacatgca
     4021 cttcaagagc gccatccacc ccagcatcct gcagaacggg ggccccatgt tcgccttccg
     4081 ccgcgtggag gagctgcaca gcaacaccga gctgggcatc gtggagtacc agcacgcctt
     4141 caagaccccc atcgccttcg ccagatctcg agctcgatga accggttggc taataaagga
     4201 aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat
     4261 atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat
     4321 atgcccatat gctggctgcc atgaacaaag gttggctata aagaggtcat cagtatatga
     4381 aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga ggttagattt
     4441 tttttatatt ttgttttgtg ttattttttt ctttaacatc cctaaaattt tccttacatg
     4501 ttttactagc cagatttttc ctcctctcct gactactccc agtcatagct gtccctcttc
     4561 tcttatggag atcggatccg aattcccgat aaggatcttc ctagagcatg gctacgtaga
     4621 taagtagcat ggcgggttaa tcattaacta caaggaaccc ctagtgatgg agttggccac
     4681 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     4741 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agccttaatt aacctaattc
     4801 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     4861 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     4921 cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt
     4981 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     5041 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     5101 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5161 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5221 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     5281 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     5341 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     5401 aacgtttata atttcaggtg gcatctttcg gggaaatgtg cgcggaaccc ctatttgttt
     5461 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5521 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5581 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5641 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaatagtgg
     5701 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5761 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5821 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5881 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5941 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     6001 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     6061 aaacgacgag cgtgacacca cgatgcctgt agtaatggta acaacgttgc gcaaactatt
     6121 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     6181 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6241 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     6301 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6361 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6421 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6481 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6541 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6601 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6661 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6721 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6781 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6841 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6901 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6961 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     7021 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7081 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7141 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7201 cttttgctgc ggttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     7261 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     7321 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     7381 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     7441 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     7501 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     7561 ctatgaccat gattacgcca gatttaatta agg
//
