LOCUS       sequence-214539-        5153 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bicistronic construct: Co-express two genes by 2A peptides.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5153)
  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 5153)
  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-Mef2c-tPT2A-GFP
FEATURES             Location/Qualifiers
     source          1..5153
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1413..1469
                     /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             1497..1550
                     /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             join(1557..1559,1560..1562,1563..2276)
                     /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(1557..1559,1560..1562,1563..2273)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1602..1623)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1863..1882)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2210..2231
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2320..2338)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2321..2338)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2356..2372)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2356..2372)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2369..2391)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2380..2396
                     /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(2404..2410,2411..2428,2429..2434))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(2585..2602)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2756..3344)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2836..2855)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3515..4306,4307..4375))
                     /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     4138..4157
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4376..4480)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4558..5013)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4695..4716)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4907..4926
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(5139..5153,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(5153,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg ggcccgacgt
       61 gatccatcga tgttaacacc ggtgacgtcg gatccaccgc catggggaga aaaaagattc
      121 agattacgag gataatggat gagcgtaaca gacaggtgac ttttacgaag aggaaatttg
      181 gattgatgaa gaaggcttat gagctgagcg tgctgtgcga ctgtgagatt gcactgatca
      241 tcttcaacag caccaacaag ctgttccagt acgccagcac tgacatggat aaggtgttgc
      301 tcaagtacac cgagtacaac gagccgcacg agagccggac aaactcagac attgtggagg
      361 cattgaacaa gaaagaaaac aaaggctctg aaagccccga tcctgactcc tcttatgcac
      421 tcaccccacg cactgaagaa aaatacaaaa aaattaatga agaatttgat aatatgatca
      481 agagtcataa aattcctgct gttccacctc ccagctttga gatgccagtt accatcccag
      541 tgtccagcca taacagtttg gtgtacagca atcctgtcag cacactggga aaccccaatc
      601 ttctgccact ggcccacccg tctctgcaga ggaatagtat gtctcctggt gtaacacata
      661 gacctccaag tgcaggtaac acaggcggtc tgatgggcgg agatctgaca tccggtgcag
      721 gcaccagcgc agggaatgga tacggcaacc cccggaactc accaggcctg ctggtctcac
      781 ctggtaacct gaacaagaat atacaagcca aatctcctcc ccctatgaat ctaggaatga
      841 ataatcgtaa gccagatctc cgcgttctta tcccacctgg cagcaagaac acgatgccat
      901 cagtgaatca aaggataaat aactcccagt cggctcagtc attggctacc ccggtggttt
      961 ccgtagcaac tcctacttta ccaggacaag gaatgggagg atatccatca gccatttcaa
     1021 caacatatgg tactgagtac tctctgagta gcgcagatct gtcatctctg tctggcttca
     1081 acactgccag tgcgctccac ctcggctctg taactggctg gcagcagcag cacctacata
     1141 acatgccgcc atctgccctc agtcagttgg gagaccgtac caccacccct tcgagatacc
     1201 cacaacacac cacgcgccac gaggcgggga ggtctcctgt tgacagcttg agcagctgta
     1261 gcagttccta cgatgggagc gaccgagagg atcaccggaa cgaattccac tcccccattg
     1321 gactcaccag accttcgccg gacgaaaggg aaagtccttc agtcaagcgc atgcgactct
     1381 ctgaaggatg ggcaacagct agcggcagcg gcgccacaaa cttctctctg ctaaagcaag
     1441 caggtgatgt tgaagaaaac cccgggccta ctagtgctgg aaagcttggc tccggcgagg
     1501 gcaggggaag tcttctaaca tgcggggacg tggaggaaaa tcccggccca ctcgagatgg
     1561 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     1621 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     1681 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     1741 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     1801 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     1861 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     1921 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     1981 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     2041 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     2101 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     2161 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     2221 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaagtcg
     2281 acgcggccgc ccaacgcgtt ggatgcatag cttgagtatt ctatagtgtc acctaaatag
     2341 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     2401 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcac
     2461 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     2521 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     2581 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     2641 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     2701 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     2761 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     2821 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     2881 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     2941 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3001 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3061 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3121 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3181 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3241 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3301 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3361 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3421 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3481 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3541 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     3601 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     3661 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     3721 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     3781 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     3841 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     3901 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     3961 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4021 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4081 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4141 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4201 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4261 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4321 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4381 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4441 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4501 cacctgatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa
     4561 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     4621 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     4681 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     4741 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     4801 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     4861 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     4921 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     4981 ccgccgcgct taatgcgccg ctacagggcg cgtccattcg ccattcaggc tgcgcaactg
     5041 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     5101 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gtt
//
