LOCUS       sequence-153009-        2834 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  P2A followed by multiple cloning site without pA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2834)
  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 2834)
  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-MCS no-pA
FEATURES             Location/Qualifiers
     source          1..2834
                     /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"
     misc_feature    95..202
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     111..127
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     112..128
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(162..178)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(162..178)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    complement(210..305)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(342..361)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(343..361)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(366..382)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(366..382)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(484..503)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             495..1304
                     /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(562..581)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1451..2039
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1940..1959
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2193..2210
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2369..2396)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2488..2574)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2623..2645
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2637..2654
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2638..2654
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(join(2725..2834,1..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 tcgaggtcga
      121 cggtatcgat aagcttgata tcgaattcct gcagcccggg ggatccacta gttctagagc
      181 ggccgccacc gcggtggagc tccagcttgc aactttatta tacaaagttg gcattataaa
      241 aaagcattgc ttatcaattt gttgcaacga acaggtcact atcagtcaaa ataaaatcat
      301 tatttggagc tccatggtag cgttaacgcg gccgcgatat cccctatagt gagtcgtatt
      361 acatggtcat agctgtttcc tggcagctct ggcccgtgtc tcaaaatctc tgatgttaca
      421 ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta
      481 atacaagggg tgttatgagc catattcaac gggaaacgtc gaggccgcga ttaaattcca
      541 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
      601 cgacaatcta tcgcttgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
      661 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
      721 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca
      781 ccactgcgat ccccggaaaa acagcattcc aggtattaga agaatatcct gattcaggtg
      841 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta
      901 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
      961 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     1021 tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     1081 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg
     1141 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
     1201 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
     1261 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa ttggttaatt
     1321 ggttgtaaca ctggcagagc attacgctga cttgacggga cggcgcaagc tcatgaccaa
     1381 aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc agaccccgta gaaaagatca
     1441 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     1501 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     1561 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     1621 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     1681 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     1741 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     1801 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gcattgagaa agcgccacgc
     1861 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     1921 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     1981 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     2041 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     2101 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     2161 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     2221 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     2281 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat acgcgtaccg
     2341 ctagccagga agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt
     2401 agtttgatgc ctggcagttt atggcgggcg tcctgcccgc caccctccgg gccgttgctt
     2461 cacaacgttc aaatccgctc ccggcggatt tgtcctactc aggagagcgt tcaccgacaa
     2521 acaacagata aaacgaaagg cccagtcttc cgactgagcc tttcgtttta tttgatgcct
     2581 ggcagttccc tactctcgcg ttaacgctag catggatgtt ttcccagtca cgacgttgta
     2641 aaacgacggc cagtcttaag ctcgggccct gcagctctag agctcgaatt ctacaggtca
     2701 ctaataccat ctaagtagtt gattcatagt gactgcatat gttgtgtttt acagtattat
     2761 gtagtctgtt ttttatgcaa aatctaattt aatatattga tatttatatc attttacgtt
     2821 tctcgttcaa cttt
//
