LOCUS       sequence-214542-        3232 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Quadcistronic construct: Co-express four genes by three 2A peptides 
            -- P2A, T2A and E2A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3232)
  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 3232)
  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-PTE2A
FEATURES             Location/Qualifiers
     source          1..3232
                     /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             117..173
                     /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             201..254
                     /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             282..341
                     /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(397..415)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(398..415)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(433..449)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(433..449)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(446..468)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    457..473
                     /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(481..487,488..505,506..511))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    526..547
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(664..681)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(835..1423)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(915..934)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1594..2385,2386..2454))
                     /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     2217..2236
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2455..2559)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2637..3092)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2774..2795)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2986..3005
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(3218..3232,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(3232,1..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 gatccgctgg agctagcggc agcggcgcca
      121 caaacttctc tctgctaaag caagcaggtg atgttgaaga aaaccccggg cctactagtg
      181 ctggaaagct tggctccggc gagggcaggg gaagtcttct aacatgcggg gacgtggagg
      241 aaaatcccgg cccactcgag ggagcatccg gaggctcggg ccagtgtact aattatgctc
      301 tcttgaaatt ggctggagat gttgagagca acccaggtcc ctctagagga gcagtcgacg
      361 cggccgccca acgcgttgga tgcatagctt gagtattcta tagtgtcacc taaatagctt
      421 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
      481 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
      541 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
      601 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
      661 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
      721 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
      781 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
      841 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
      901 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
      961 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     1021 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     1081 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     1141 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     1201 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     1261 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     1321 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     1381 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     1441 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     1501 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     1561 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     1621 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     1681 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     1741 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     1801 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     1861 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     1921 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     1981 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     2041 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     2101 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     2161 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     2221 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     2281 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     2341 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     2401 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     2461 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     2521 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     2581 acctgatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat
     2641 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     2701 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     2761 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     2821 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     2881 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     2941 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     3001 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     3061 cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc cattcaggct gcgcaactgt
     3121 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     3181 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg tt
//
