LOCUS       sequence-375242-        4992 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP  KIKO.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4992)
  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 4992)
  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"; doi: 
            "10.1038/s41592-019-0329-7"; journalName: "Nat Methods"; date: 
            "2019-03"; volume: "16"; issue: "3"; pages: "247-254"
COMMENT     Sequence Label: pXD053 GFP KIKO
FEATURES             Location/Qualifiers
     source          1..4992
                     /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        880..1091
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1111..1120
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1117..1119,1120..1122,1123..1836)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1117..1119,1120..1122,1123..1833)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1162..1183)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1423..1442)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1770..1791
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1843..1891
                     /note="synthetic polyadenylation signal"
     repeat_region   2255..2395
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2255..2384
                     /label=AAV2 ITR
     rep_origin      2470..2925
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2557..2576)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2767..2788
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2942..2961)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3061..3083
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3121..3139)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3207..3311
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3312..3380,3381..4172)
                     /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(3530..3549)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4343..4931
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4832..4851
                     /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 atgagtctct cagctggtac actaatttct actaagtgta gatgcacgaa gctctccgat
      481 gtgtaatttc tactaagtgt agattttttt aagcttggcg tggatccgat atcaactaga
      541 tcttgagaca aggtacgtaa ccacgtgggc agcctcacgt agaaggaaga ggctctgcag
      601 tggaggccag tgcccatccc cgggtggcag aggccccagc agagacttct caatgacatt
      661 ccagctgggg tggcccttcc agagcccttg ctgcccgagg gatgtgagca ggtggccggg
      721 gaggctttgt ggggccaccc agccccttcc tcacctctct ccatctctca gactccccag
      781 acaggccctg gaaccccccc accttctccc cagccctgct cgtggtgacc gaaggggaca
      841 acgccacctt cacctgcagc ttctccaaca cagttaacag ggcagagcgc acatcgccca
      901 cagtccccga gaagttgggg ggaggggtcg gcaattgatc cggtgcctag agaaggtggc
      961 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
     1021 gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg
     1081 ccagaacaca ggaccggttc tagacgtacg gccaccatgg tgagcaaggg cgaggagctg
     1141 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     1201 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     1261 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     1321 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     1381 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     1441 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     1501 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     1561 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     1621 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     1681 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     1741 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     1801 gggatcactc tcggcatgga cgagctgtac aagtaagcta gcaataaaag atctttattt
     1861 tcattagatc tgtgtgttgg ttttttgtgt gaagaattct cggagagctt cgtgctaaac
     1921 tggtaccgca tgagccccag caaccagacg gacaagctgg ccgccttccc cgaggaccgc
     1981 agccagcccg gccaggactg ccgcttccgt gtcacacaac tgcccaacgg gcgtgacttc
     2041 cacatgagcg tggtcagggc ccggcgcaat gacagcggca cctacctctg tggggccatc
     2101 tccctggccc ccaaggcgca gatcaaagag agcctgcggg cagagctcag ggtgacaggt
     2161 gcggcctcgg aggccccggg gcaggggtga gctgagccgg tcctggggtg ggtgtcccct
     2221 cctgcacagg atcaggtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc
     2281 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     2341 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc
     2401 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     2461 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2521 gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct
     2581 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2641 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     2701 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2761 ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt
     2821 ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac
     2881 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     2941 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     3001 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     3061 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     3121 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     3181 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3241 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3301 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     3361 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     3421 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     3481 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     3541 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     3601 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     3661 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     3721 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     3781 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     3841 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     3901 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     3961 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     4021 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     4081 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     4141 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     4201 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     4261 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4321 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4381 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4441 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     4501 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4561 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4621 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4681 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4741 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     4801 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     4861 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     4921 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4981 ttgctcacat gt
//
