LOCUS       sequence-153011-        3539 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  P2A followed by GFP without pA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3539)
  AUTHORS   Fowler DK, Stewart S, Seredick S, Eisen JS, Stankunas K, Washbourne 
            P
  TITLE     A MultiSite Gateway Toolkit for Rapid Cloning of Vertebrate 
            Expression Constructs with Diverse Research Applications.
  JOURNAL   PLoS One. 2016 Aug 8;11(8):e0159277. doi: 
            10.1371/journal.pone.0159277. eCollection 2016.
  PUBMED    27500400
REFERENCE   2  (bases 1 to 3539)
  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: "PLoS One.";
            date: "2016-08-8"; volume: "11(8):e0159277. doi"; pages: " 
            10.1371/journal.pone.0159277. eCollection 2016"
COMMENT     Sequence Label: p3E-P2A-GFP no-pA
FEATURES             Location/Qualifiers
     source          1..3539
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             38..94
                     /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             join(116..118,119..121,122..835)
                     /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(116..118,119..121,122..832)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(161..182)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(422..441)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     769..790
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(867..883)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(867..883)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    complement(915..1010)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1047..1066)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1048..1066)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1071..1087)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1071..1087)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1189..1208)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1200..2009
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1267..1286)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2156..2744
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2645..2664
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2898..2915
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3074..3101)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3193..3279)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3328..3350
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3342..3359
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3343..3359
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(join(3430..3539,1..14))
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 cttgtacaaa gtggaagaat tgggtaccgg atccggagcc acgaacttct ctctgttaaa
       61 gcaagcagga gacgtggaag aaaaccccgg tcctggtacc gggccccccc tcgagatggt
      121 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
      181 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
      241 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
      301 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
      361 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
      421 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
      481 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
      541 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
      601 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
      661 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
      721 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
      781 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaaaagct
      841 tgatatcgaa ttcctgcagc ccgggggatc cactagttct agagcggccg ccaccgcggt
      901 ggagctccag cttgcaactt tattatacaa agttggcatt ataaaaaagc attgcttatc
      961 aatttgttgc aacgaacagg tcactatcag tcaaaataaa atcattattt ggagctccat
     1021 ggtagcgtta acgcggccgc gatatcccct atagtgagtc gtattacatg gtcatagctg
     1081 tttcctggca gctctggccc gtgtctcaaa atctctgatg ttacattgca caagataaaa
     1141 atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta
     1201 tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt
     1261 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgct
     1321 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca
     1381 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga
     1441 ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg
     1501 gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg
     1561 cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca
     1621 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg
     1681 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc
     1741 ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata
     1801 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg
     1861 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat
     1921 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt
     1981 ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg taacactggc
     2041 agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc cttaacgtga
     2101 gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2161 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2221 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     2281 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     2341 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     2401 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     2461 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     2521 ccgaactgag atacctacag cgtgagcatt gagaaagcgc cacgcttccc gaagggagaa
     2581 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2641 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2701 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2761 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2821 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     2881 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     2941 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     3001 actggaaagc gggcagtgag cgcaacgcaa ttaatacgcg taccgctagc caggaagagt
     3061 ttgtagaaac gcaaaaaggc catccgtcag gatggccttc tgcttagttt gatgcctggc
     3121 agtttatggc gggcgtcctg cccgccaccc tccgggccgt tgcttcacaa cgttcaaatc
     3181 cgctcccggc ggatttgtcc tactcaggag agcgttcacc gacaaacaac agataaaacg
     3241 aaaggcccag tcttccgact gagcctttcg ttttatttga tgcctggcag ttccctactc
     3301 tcgcgttaac gctagcatgg atgttttccc agtcacgacg ttgtaaaacg acggccagtc
     3361 ttaagctcgg gccctgcagc tctagagctc gaattctaca ggtcactaat accatctaag
     3421 tagttgattc atagtgactg catatgttgt gttttacagt attatgtagt ctgtttttta
     3481 tgcaaaatct aatttaatat attgatattt atatcatttt acgtttctcg ttcaacttt
//
