LOCUS       sequence-375241-        7149 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dTomato GFP KIKO.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7149)
  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 7149)
  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: pXD050 dTomato GFP KIKO
FEATURES             Location/Qualifiers
     source          1..7149
                     /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"
     promoter        3037..3248
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3268..3277
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3274..3276,3277..3279,3280..3993)
                     /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(3274..3276,3277..3279,3280..3990)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3319..3340)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3580..3599)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3927..3948
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4000..4048
                     /note="synthetic polyadenylation signal"
     repeat_region   4412..4552
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4412..4541
                     /label=AAV2 ITR
     rep_origin      4627..5082
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4714..4733)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4924..4945
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5099..5118)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5218..5240
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5278..5296)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5364..5468
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5469..5537,5538..6329)
                     /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(5687..5706)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6500..7088
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6989..7008
                     /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 cgtgggcagc ctcacgtaga aggaagaggc tctgcagtgg
     2761 aggccagtgc ccatccccgg gtggcagagg ccccagcaga gacttctcaa tgacattcca
     2821 gctggggtgg cccttccaga gcccttgctg cccgagggat gtgagcaggt ggccggggag
     2881 gctttgtggg gccacccagc cccttcctca cctctctcca tctctcagac tccccagaca
     2941 ggccctggaa cccccccacc ttctccccag ccctgctcgt ggtgaccgaa ggggacaacg
     3001 ccaccttcac ctgcagcttc tccaacacag ttaacagggc agagcgcaca tcgcccacag
     3061 tccccgagaa gttgggggga ggggtcggca attgatccgg tgcctagaga aggtggcgcg
     3121 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
     3181 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca
     3241 gaacacagga ccggttctag acgtacggcc accatggtga gcaagggcga ggagctgttc
     3301 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     3361 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     3421 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     3481 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     3541 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     3601 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     3661 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     3721 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     3781 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     3841 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     3901 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     3961 atcactctcg gcatggacga gctgtacaag taagctagca ataaaagatc tttattttca
     4021 ttagatctgt gtgttggttt tttgtgtgaa gaattctcgg agagcttcgt gctaaactgg
     4081 taccgcatga gccccagcaa ccagacggac aagctggccg ccttccccga ggaccgcagc
     4141 cagcccggcc aggactgccg cttccgtgtc acacaactgc ccaacgggcg tgacttccac
     4201 atgagcgtgg tcagggcccg gcgcaatgac agcggcacct acctctgtgg ggccatctcc
     4261 ctggccccca aggcgcagat caaagagagc ctgcgggcag agctcagggt gacaggtgcg
     4321 gcctcggagg ccccggggca ggggtgagct gagccggtcc tggggtgggt gtcccctcct
     4381 gcacaggatc aggtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact
     4441 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     4501 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
     4561 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     4621 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     4681 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc
     4741 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     4801 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     4861 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4921 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
     4981 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
     5041 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     5101 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     5161 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     5221 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     5281 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     5341 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     5401 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     5461 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     5521 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     5581 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     5641 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     5701 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     5761 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     5821 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     5881 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     5941 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     6001 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     6061 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     6121 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     6181 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     6241 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     6301 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     6361 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     6421 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     6481 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6541 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6601 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     6661 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6721 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6781 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6841 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     6901 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     6961 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     7021 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     7081 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     7141 ctcacatgt
//
