LOCUS       sequence-209944-        5127 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 5127)
  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 5127)
  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..5127
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1345..1401
                     /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             1429..1482
                     /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(1489..1491,1492..1494,1495..2208)
                     /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(1489..1491,1492..1494,1495..2205)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1534..1555)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1795..1814)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2142..2163
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2252..2270)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2253..2270)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2288..2304)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2288..2304)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2301..2323)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2312..2328
                     /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(2336..2342,2343..2360,2361..2366))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2381..2402
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2519..2536)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2690..3278)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2770..2789)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3449..4240,4241..4309))
                     /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     4072..4091
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4310..4414)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4492..4947)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4629..4650)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4841..4860
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5073..5095
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5087..5104
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5088..5104
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5111..5127,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5111..5127,1..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 ctagcggcag cggcgccaca aacttctctc tgctaaagca agcaggtgat
     1381 gttgaagaaa accccgggcc tactagtgct ggaaagcttg gctccggcga gggcagggga
     1441 agtcttctaa catgcgggga cgtggaggaa aatcccggcc cactcgagat ggtgagcaag
     1501 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1561 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1621 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1681 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     1741 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     1801 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1861 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1921 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     1981 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     2041 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     2101 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2161 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaagt cgacgcggcc
     2221 gcccaacgcg ttggatgcat agcttgagta ttctatagtg tcacctaaat agcttggcgt
     2281 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     2341 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     2401 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     2461 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     2521 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     2581 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2641 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2701 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2761 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2821 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     2881 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     2941 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3001 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3061 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3121 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3181 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3241 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3301 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3361 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3421 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     3481 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     3541 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     3601 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3661 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3721 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     3781 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     3841 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     3901 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     3961 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4021 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4081 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4141 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4201 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4261 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4321 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4381 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     4441 atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gaaattgtaa
     4501 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     4561 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     4621 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     4681 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     4741 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     4801 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     4861 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     4921 cgcttaatgc gccgctacag ggcgcgtcca ttcgccattc aggctgcgca actgttggga
     4981 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
     5041 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc
     5101 cagtgaattg taatacgact cactata
//
