LOCUS       sequence-375244-        5743 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dTomato  KIKO.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5743)
  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 5743)
  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: pXD042 dTomato KIKO
FEATURES             Location/Qualifiers
     source          1..5743
                     /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        1252..1463
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1483..1492
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1489..2190
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(1622..1640)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1890..1909
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2084..2107
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    2200..2248
                     /note="synthetic polyadenylation signal"
     polyA_signal    2930..2978
                     /note="synthetic polyadenylation signal"
     repeat_region   3006..3146
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3006..3135
                     /label=AAV2 ITR
     rep_origin      3221..3676
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3308..3327)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3518..3539
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3693..3712)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3812..3834
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3872..3890)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3958..4062
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4063..4131,4132..4923)
                     /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(4281..4300)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5094..5682
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5583..5602
                     /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 aggtacgatg taaggagctg ctgtgacttg ctcaaggcct tatatcgagt
      601 aaacggtagc gctggggctt agacgcaggt gttctgattt atagttcaaa acctctatca
      661 atgagagagc aatctcctgg taatgtgata gatttcccaa cttaatgcca acataccata
      721 aacctcccat tctgctaatg cccagcctaa gttggggaga ccactccaga tttcaagatg
      781 tacagtttgc tttgctgggc ctttttccca tgcctgcctt tactctgcca gagttatatt
      841 gctggggttt tgaagaagat cctattaaat aaaagaataa gcagtattat taagtagccc
      901 tgcatttcag gtttccttga gtggcaggcc aggcctggcc gtgaacgttc actgaaatca
      961 tggcctcttg gccaagattg atagcttgtg cctgtccctg agtcccagtc cgtcacgagc
     1021 agctggtttc taatatgcta tttcccgtat aaagcatgag accgtgactt gccagcccca
     1081 cagagccccg cccttgtcca tcactggcat ctggactcca gcctgggttg gggcaaagag
     1141 ggaaatgaga tcatgtccta accctgatcc tcttgtccca cagatatcca gaaccctgac
     1201 ccagcgctta ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag tgggcagagc
     1261 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga tccggtgcct
     1321 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc
     1381 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca
     1441 acgggtttgc cgccagaaca caggaccggt tctagacgta cggccaccat ggtgagcaag
     1501 ggcgaggagg tcatcaaaga gttcatgcgc ttcaaggtgc gcatggaggg ctccatgaac
     1561 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
     1621 gccaagctga aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtccccc
     1681 cagttcatgt acggctccaa ggcgtacgtg aagcaccccg ccgacatccc cgattacaag
     1741 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggt
     1801 ctggtgaccg tgacccagga ctcctccctg caggacggca cgctgatcta caaggtgaag
     1861 atgcgcggca ccaacttccc ccccgacggc cccgtaatgc agaagaagac catgggctgg
     1921 gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc tgaagggcga gatccaccag
     1981 gccctgaagc tgaaggacgg cggccactac ctggtggagt tcaagaccat ctacatggcc
     2041 aagaagcccg tgcaactgcc cggctactac tacgtggaca ccaagctgga catcacctcc
     2101 cacaacgagg actacaccat cgtggaacag tacgagcgct ccgagggccg ccaccacctg
     2161 ttcctgtacg gcatggacga gctgtacaag taagctagca ataaaagatc tttattttca
     2221 ttagatctgt gtgttggttt tttgtgtggt accgagagac tctaaatcca gtgacaagtc
     2281 tgtctgccta ttcaccgatt ttgattctca aacaaatgtg tcacaaagta aggattctga
     2341 tgtgtatatc acagacaaaa ctgtgctaga catgaggtct atggacttca agagcaacag
     2401 tgctgtggcc tggagcaaca aatctgactt tgcatgtgca aacgccttca acaacagcat
     2461 tattccagca gacaccttct tccccagccc aggtaagggc agctttggtg ccttcgcagg
     2521 ctgtttcctt gcttcaggaa tggccaggtt ctgcccagag ctctggtcaa tgatgtctaa
     2581 aactcctctg attggtggtc tcggccttat ccattgccac caaaaccctc tttttactaa
     2641 gaaacagtga gccttgttct ggcagtccag agaatgacac ggaaaaaaag cagatgaaga
     2701 gaaggtggca ggagagggca cgtggcccag cctcagtctc tccaactgag ttcctgcctg
     2761 cctgcctttg ctcagactgt ttgcccctta ctgctcttct aggcctcatt ctaagcccct
     2821 tctccaagtt gcctctcctt atttctccct gtctgccaaa aaatctttcc cagctcacta
     2881 agtcagtctc acgcagtcac tcattaaccc acaattcgat atcaagctta ataaaagatc
     2941 tttattttca ttagatctgt gtgttggttt tttgtgtggt aaccacgtgc ggaccgagcg
     3001 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     3061 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     3121 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     3181 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     3241 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     3301 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     3361 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     3421 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     3481 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     3541 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     3601 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     3661 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     3721 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3781 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3841 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     3901 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     3961 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     4021 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     4081 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     4141 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     4201 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     4261 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     4321 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     4381 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     4441 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     4501 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     4561 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     4621 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     4681 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     4741 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     4801 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     4861 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     4921 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     4981 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     5041 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     5101 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5161 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     5221 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     5281 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5341 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     5401 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     5461 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     5521 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     5581 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     5641 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     5701 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
