LOCUS       sequence-210073-        8790 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Quadcistronic construct: Co-express four genes by three 2A peptides 
            -- T2A, P2A and E2A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8790)
  AUTHORS   Liu Z, Chen O, Wall JBJ, Zheng M, Zhou Y, Wang L, Ruth Vaseghi H, 
            Qian L, Liu J
  TITLE     Systematic comparison of 2A peptides for cloning multi-genes in a 
            polycistronic vector.
  JOURNAL   Sci Rep. 2017 May 19;7(1):2193. doi: 10.1038/s41598-017-02460-2.
  PUBMED    28526819
REFERENCE   2  (bases 1 to 8790)
  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"; journalName: "Sci Rep."; 
            date: "2017-05-19"; volume: "7(1):2193. doi"; pages: " 
            10.1038/s41598-017-02460-2"
COMMENT     Sequence Label: pGEMT-TPE2A-Mef2c-Tdtomato-Gata4-Tbx5
FEATURES             Location/Qualifiers
     source          1..8790
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1345..1398
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1405..2832
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGRYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     CDS             2848..2904
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4249..4308
                     /codon_start=1
                     /product="2A peptide from equine rhinitis A virus 
                     polyprotein"
                     /label=E2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="QCTNYALLKLAGDVESNPGP"
     promoter        complement(5915..5933)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5916..5933)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5951..5967)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5951..5967)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5964..5986)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5975..5991
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5999..6005,6006..6023,6024..6029))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6044..6065
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6182..6199)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6353..6941)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6433..6452)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7112..7903,7904..7972))
                     /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     7735..7754
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7973..8077)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(8155..8610)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8292..8313)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8504..8523
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8736..8758
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8750..8767
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8751..8767
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(8774..8790,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(8774..8790,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgacgt cggatccacc gccatgggga gaaaaaagat tcagattacg
       61 aggataatgg atgagcgtaa cagacaggtg acttttacga agaggaaatt tggattgatg
      121 aagaaggctt atgagctgag cgtgctgtgc gactgtgaga ttgcactgat catcttcaac
      181 agcaccaaca agctgttcca gtacgccagc actgacatgg ataaggtgtt gctcaagtac
      241 accgagtaca acgagccgca cgagagccgg acaaactcag acattgtgga ggcattgaac
      301 aagaaagaaa acaaaggctc tgaaagcccc gatcctgact cctcttatgc actcacccca
      361 cgcactgaag aaaaatacaa aaaaattaat gaagaatttg ataatatgat caagagtcat
      421 aaaattcctg ctgttccacc tcccagcttt gagatgccag ttaccatccc agtgtccagc
      481 cataacagtt tggtgtacag caatcctgtc agcacactgg gaaaccccaa tcttctgcca
      541 ctggcccacc cgtctctgca gaggaatagt atgtctcctg gtgtaacaca tagacctcca
      601 agtgcaggta acacaggcgg tctgatgggc ggagatctga catccggtgc aggcaccagc
      661 gcagggaatg gatacggcaa cccccggaac tcaccaggcc tgctggtctc acctggtaac
      721 ctgaacaaga atatacaagc caaatctcct ccccctatga atctaggaat gaataatcgt
      781 aagccagatc tccgcgttct tatcccacct ggcagcaaga acacgatgcc atcagtgaat
      841 caaaggataa ataactccca gtcggctcag tcattggcta ccccggtggt ttccgtagca
      901 actcctactt taccaggaca aggaatggga ggatatccat cagccatttc aacaacatat
      961 ggtactgagt actctctgag tagcgcagat ctgtcatctc tgtctggctt caacactgcc
     1021 agtgcgctcc acctcggctc tgtaactggc tggcagcagc agcacctaca taacatgccg
     1081 ccatctgccc tcagtcagtt gggagaccgt accaccaccc cttcgagata cccacaacac
     1141 accacgcgcc acgaggcggg gaggtctcct gttgacagct tgagcagctg tagcagttcc
     1201 tacgatggga gcgaccgaga ggatcaccgg aacgaattcc actcccccat tggactcacc
     1261 agaccttcgc cggacgaaag ggaaagtcct tcagtcaagc gcatgcgact ctctgaagga
     1321 tgggcaacag ctagcggctc cggcgagggc aggggaagtc ttctaacatg cggggacgtg
     1381 gaggaaaatc ccggcccaac tagtatggtg agcaagggcg aggaggtcat caaagagttc
     1441 atgcgcttca aggtgcgcat ggagggctcc atgaacggcc acgagttcga gatcgagggc
     1501 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggc
     1561 ggccccctgc ccttcgcctg ggacatcctg tccccccagt tcatgtacgg ctccaaggcg
     1621 tacgtgaagc accccgccga catccccgat tacaagaagc tgtccttccc cgagggcttc
     1681 aagtgggagc gcgtgatgaa cttcgaggac ggcggtctgg tgaccgtgac ccaggactcc
     1741 tccctgcagg acggcacgct gatctacaag gtgaagatgc gcggcaccaa cttccccccc
     1801 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctccaccga gcgcctgtac
     1861 ccccgcgacg gcgtgctgaa gggcgagatc caccaggccc tgaagctgaa ggacggcggc
     1921 cactacctgg tggagttcaa gaccatctac atggccaaga agcccgtgca actgcccggc
     1981 tactactacg tggacaccaa gctggacatc acctcccaca acgaggacta caccatcgtg
     2041 gaacagtacg agcgctccga gggccgccac cacctgttcc tggggcatgg caccggcagc
     2101 accggcagcg gcagctccgg caccgcctcc tccgaggaca acaacatggc cgtcatcaaa
     2161 gagttcatgc gcttcaaggt gcgcatggag ggctccatga acggccacga gttcgagatc
     2221 gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc
     2281 aagggcggcc ccctgccctt cgcctgggac atcctgtccc cccagttcat gtacggctcc
     2341 aaggcgtacg tgaagcaccc cgccgacatc cccgattaca agaagctgtc cttccccgag
     2401 ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gtctggtgac cgtgacccag
     2461 gactcctccc tgcaggacgg cacgctgatc tacaaggtga agatgcgcgg caccaacttc
     2521 ccccccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc caccgagcgc
     2581 ctgtaccccc gcgacggcgt gctgaagggc gagatccacc aggccctgaa gctgaaggac
     2641 ggcggccgct acctggtgga gttcaagacc atctacatgg ccaagaagcc cgtgcaactg
     2701 cccggctact actacgtgga caccaagctg gacatcacct cccacaacga ggactacacc
     2761 atcgtggaac agtacgagcg ctccgagggc cgccaccacc tgttcctgta cggcatggac
     2821 gagctgtaca agaagcttgg cagcggcgcc acaaacttct ctctgctaaa gcaagcaggt
     2881 gatgttgaag aaaaccccgg gcctctcgag atgtaccaaa gcctggccat ggccgccaac
     2941 cacggccccc cgcccggcgc ctacgaagca ggtggccctg gcgccttcat gcacagcgcg
     3001 ggcgccgcgt cctcgcccgt ctacgtgccc actccgcggg tgccgtcctc tgtgctgggc
     3061 ctgtcctacc tgcagggcgg tggcagtgcc gctgcagctg gaaccacctc gggtggcagc
     3121 tccggggccg gcccgtcggg tgcagggcct gggacccagc agggtagccc tggctggagc
     3181 caagctggag ccgagggagc cgcctacacc ccgccgcccg tgtccccgcg cttctctttc
     3241 ccggggacta ctgggtccct ggcagcagct gccgccgctg ccgcagcccg ggaagctgca
     3301 gcctacggca gtggcggcgg ggcggcgggc gctggtctgg ctggccgaga gcagtacggg
     3361 cgtccgggct tcgccggctc ctactccagc ccctacccag cctacatggc cgacgtggga
     3421 gcatcctggg ccgcagccgc tgccgcctct gccggcccct tcgacagccc agtcctgcac
     3481 agcctgcctg gacgggccaa ccctggaaga caccccaatc tcgatatgtt tgatgacttc
     3541 tcagaaggca gagagtgtgt caattgtggg gccatgtcca ccccactctg gaggcgagat
     3601 gggacgggac actacctgtg caatgcctgt ggcctctatc acaagatgaa cggcatcaac
     3661 cggcccctca ttaagcctca gcgccgcctg tccgcttccc gccgggtagg cctctcctgt
     3721 gccaactgcc agactaccac caccacgctg tggcgtcgta atgccgaggg tgagcctgta
     3781 tgtaatgcct gcggcctcta catgaagctc catggggttc ccaggcctct tgcaatgcgg
     3841 aaggagggga ttcaaaccag aaaacggaag cccaagaacc tgaataaatc taagacgcca
     3901 gcaggtcctg ctggtgagac cctccctccc tccagtggtg cctccagcgg taactccagc
     3961 aatgccacta gcagcagcag cagcagtgaa gagatgcgcc ccatcaagac agagcccggg
     4021 ctgtcatctc actatgggca cagcagctcc atgtcccaga cattcagtac tgtgtccggc
     4081 cacgggccct ccatccatcc agtgctgtct gctctgaagc tgtccccaca aggctatgca
     4141 tctcctgtca ctcagacatc gcaggccagc tccaagcagg actcttggaa cagcctggtc
     4201 ctggctgaca gtcatgggga cataatcacc gcgtccggag gctcgggcca gtgtactaat
     4261 tatgctctct tgaaattggc tggagatgtt gagagcaacc caggtccctc tagaatggcc
     4321 gatacagatg agggctttgg cctggcgcgc acgcctctgg agcctgattc caaagacagg
     4381 tcttgcgatt cgaaacctga gagtgctctg ggggctccca gcaagtctcc atcatccccg
     4441 caggctgcct tcacccagca gggcatggaa ggaatcaagg tgtttcttca tgaacgtgaa
     4501 ctgtggctga agttccacga agtgggcaca gagatgatca tcaccaaggc agggaggaga
     4561 atgtttccta gttacaaagt gaaggtgact ggccttaatc ccaaaacgaa gtatattctt
     4621 ctcatggata ttgttcccgc agacgaccac agatataaat ttgctgataa caaatggtcc
     4681 gtaactggca aagcagagcc tgccatgccg gggcgccttt acgtgcaccc ggactcccca
     4741 gcaaccggag cccactggat gcgacaactt gtctccttcc agaagctcaa actcaccaac
     4801 aaccacctgg acccgtttgg acacattatc ctgaactcca tgcacaaata ccagccccga
     4861 ttacacatcg tgaaagcaga cgaaaataat gggttcggtt caaagaacac tgcgttttgc
     4921 acccacgtct tcccggagac agcttttatc gctgtgactt cgtaccagaa tcacaagatc
     4981 acacagctga aaattgagaa caaccccttc gccaaaggct ttcggggcag tgatgacctg
     5041 gagttacaca ggatgtctcg gatgcaaagt aaagagtatc ctgtggttcc caggagcaca
     5101 gtgaggcaca aagtcacctc caaccacagc cccttcagca gcgagacccg agctctctcc
     5161 acctcatcca atttagggtc ccagtaccag tgtgagaatg gtgtctctgg cccctcccag
     5221 gaccttctgc ccccacctaa cccataccca ctggcccagg agcacagcca aatttaccac
     5281 tgtaccaaga ggaaagatga ggaatgttcc agcacggagc acccctataa gaagccgtac
     5341 atggagacat cccccagcga ggaagacacc ttctatcgct cgggctaccc ccagcagcag
     5401 ggcctgagta cctcttacag gacagagtcg gcccagcggc aggcctgcat gtatgccagc
     5461 tccgctcccc ccagcgagcc cgtgcctagc ctggaggaca tcagctgtaa cacatggccc
     5521 agcatgccct cctatagcag ctgtaccgtc accaccgtgc agcccatgga ccgtcttccc
     5581 taccagcact tctccgctca tttcacctcg gggcccctgg tccctcggtt ggctggcatg
     5641 gccaaccatg gttctcccca gctcggcgaa gggatgtttc agcaccagac ctcagtggcc
     5701 catcagcctg tggtcaggca gtgcgggcct cagactggcc ttcagtctcc gggcggcctc
     5761 cagcccccag agtttctcta cactcacggc gtgcccagga ccctgtcccc ccatcagtat
     5821 cactcggtac acggcgtcgg catggtgcca gagtggagtg agaatagcta agtcgacgcg
     5881 gccgcccaac gcgttggatg catagcttga gtattctata gtgtcaccta aatagcttgg
     5941 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     6001 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     6061 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     6121 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     6181 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     6241 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     6301 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     6361 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     6421 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6481 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6541 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6601 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6661 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6721 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6781 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6841 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6901 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6961 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     7021 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     7081 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     7141 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     7201 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     7261 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     7321 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     7381 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     7441 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     7501 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     7561 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7621 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7681 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     7741 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7801 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7861 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7921 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7981 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     8041 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     8101 ctgatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggaaattg
     8161 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     8221 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     8281 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     8341 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     8401 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     8461 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     8521 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     8581 ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc gcaactgttg
     8641 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     8701 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     8761 ggccagtgaa ttgtaatacg actcactata
//
