LOCUS       sequence-375239-        4371 bp    DNA     circular SYN 14-APR-2025
DEFINITION  KIKO AAV vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4371)
  AUTHORS   Dai X, Park JJ, Du Y, Kim HR, Wang G, Errami Y, Chen S
  TITLE     One-step generation of modular CAR-T cells with AAV-Cpf1.
  JOURNAL   Nat Methods. 2019 Mar;16(3):247-254. doi: 10.1038/s41592-019-0329-7.
            Epub 2019 Feb 25.
  PUBMED    30804551
REFERENCE   2  (bases 1 to 4371)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41592-019-0329-7"; journalName: "Nat Methods"; date: 
            "2019-03"; volume: "16"; issue: "3"; pages: "247-254"
COMMENT     Sequence Label: pXD017 KIKO AAV vector
FEATURES             Location/Qualifiers
     source          1..4371
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        152..400
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     152..172
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     323..342
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        535..746
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      766..775
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(772..774,775..777,778..1488)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(772..774,775..777,778..1488)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(817..838)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1078..1097)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1425..1446
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1489..1536
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    1558..1606
                     /note="synthetic polyadenylation signal"
     repeat_region   1634..1774
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1634..1763
                     /label=AAV2 ITR
     rep_origin      1849..2304
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1936..1955)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2146..2167
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2321..2340)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2440..2462
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2500..2518)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2586..2690
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2691..2759,2760..3551)
                     /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"
     primer_bind     complement(2909..2928)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3722..4310
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4211..4230
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgttctaga agagggccta tttcccatga ttccttcata
      181 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
      241 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
      301 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
      361 tttcttggct ttatatatct tgtggaaagg acgaaacacc gtaatttcta ctaagtgtag
      421 atggtcttcg agaagacctt ttttaagctt ggcgtggatc cgatatcaac tagatcttga
      481 gacaaggtac ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt cagtgggcag
      541 agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgatccggtg
      601 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt
      661 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc
      721 gcaacgggtt tgccgccaga acacaggacc ggttctagac gtacggccac catggtgagc
      781 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
      841 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
      901 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
      961 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     1021 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     1081 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1141 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1201 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     1261 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     1321 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     1381 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     1441 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagaa gcgtcctgct
     1501 gctactaaga aagctggtca agctaagaaa aagaaataag aattcgatat caagcttaat
     1561 aaaagatctt tattttcatt agatctgtgt gttggttttt tgtgtggtaa ccacgtgcgg
     1621 accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     1681 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     1741 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     1801 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     1861 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     1921 gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1981 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2041 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     2101 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2161 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc
     2221 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     2281 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     2341 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     2401 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     2461 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     2521 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     2581 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2641 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     2701 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     2761 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     2821 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     2881 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     2941 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     3001 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     3061 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     3121 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     3181 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     3241 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     3301 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     3361 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     3421 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     3481 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     3541 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     3601 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     3661 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     3721 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     3781 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3841 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     3901 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3961 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4021 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4081 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4141 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4201 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4261 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4321 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
