LOCUS       sequence-375246-        4437 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TRAC PDCD1 AAV double KO AAV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4437)
  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 4437)
  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: pXD017-39 TRAC PDCD1 AAV double KO AAV
FEATURES             Location/Qualifiers
     source          1..4437
                     /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        601..812
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      832..841
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(838..840,841..843,844..1554)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(838..840,841..843,844..1554)
                     /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(883..904)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1144..1163)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1491..1512
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1555..1602
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    1624..1672
                     /note="synthetic polyadenylation signal"
     repeat_region   1700..1840
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1700..1829
                     /label=AAV2 ITR
     rep_origin      1915..2370
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2002..2021)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2212..2233
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2387..2406)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2506..2528
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2566..2584)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2652..2756
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2757..2825,2826..3617)
                     /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(2975..2994)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3788..4376
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4277..4296
                     /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 aggtacctag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt
      601 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgat
      661 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
      721 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
      781 tttttcgcaa cgggtttgcc gccagaacac aggaccggtt ctagacgtac ggccaccatg
      841 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
      901 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
      961 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1021 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     1081 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     1141 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1201 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1261 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     1321 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     1381 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     1441 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1501 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagaagcgt
     1561 cctgctgcta ctaagaaagc tggtcaagct aagaaaaaga aataagaatt cgatatcaag
     1621 cttaataaaa gatctttatt ttcattagat ctgtgtgttg gttttttgtg tggtaaccac
     1681 gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc
     1741 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg
     1801 gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt
     1861 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
     1921 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     1981 ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     2041 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     2101 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
     2161 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     2221 tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga
     2281 ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     2341 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
     2401 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     2461 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     2521 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     2581 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     2641 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     2701 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     2761 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     2821 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     2881 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     2941 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     3001 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     3061 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     3121 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     3181 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     3241 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     3301 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     3361 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     3421 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     3481 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     3541 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     3601 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     3661 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     3721 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     3781 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     3841 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     3901 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     3961 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4021 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4081 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4141 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4201 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4261 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4321 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     4381 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
