LOCUS       sequence-153008-        3518 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  P2A followed by mKate2 without pA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3518)
  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 3518)
  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-mKate2 no-pA
FEATURES             Location/Qualifiers
     source          1..3518
                     /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             116..814
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     primer_bind     complement(846..862)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(846..862)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    complement(894..989)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1026..1045)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1027..1045)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1050..1066)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1050..1066)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1168..1187)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1179..1988
                     /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(1246..1265)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2135..2723
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2624..2643
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2877..2894
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3053..3080)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3172..3258)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3307..3329
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3321..3338
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3322..3338
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(join(3409..3518,1..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 gagcgagctg attaaggaga acatgcacat gaagctgtac atggagggca ccgtgaacaa
      181 ccaccacttc aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat
      241 gagaatcaag gcggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctaccag
      301 cttcatgtac ggcagcaaaa ccttcatcaa ccacacccag ggcatccccg acttctttaa
      361 gcagtccttc cccgagggct tcacatggga gagagtcacc acatacgaag acgggggcgt
      421 gctgaccgct acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat
      481 cagaggggtg aacttcccat ccaacggccc tgtgatgcag aagaaaacac tcggctggga
      541 ggcctccacc gagaccctgt accccgctga cggcggcctg gaaggcagag ccgacatggc
      601 cctgaagctc gtgggcgggg gccacctgat ctgcaacttg aagaccacat acagatccaa
      661 gaaacccgct aagaacctca agatgcccgg cgtctactat gtggacagaa gactggaaag
      721 aatcaaggag gccgacaaag agacctacgt cgagcagcac gaggtggctg tggccagata
      781 ctgcgacctc cctagcaaac tggggcacag atgaaagctt gatatcgaat tcctgcagcc
      841 cgggggatcc actagttcta gagcggccgc caccgcggtg gagctccagc ttgcaacttt
      901 attatacaaa gttggcatta taaaaaagca ttgcttatca atttgttgca acgaacaggt
      961 cactatcagt caaaataaaa tcattatttg gagctccatg gtagcgttaa cgcggccgcg
     1021 atatccccta tagtgagtcg tattacatgg tcatagctgt ttcctggcag ctctggcccg
     1081 tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca tgaacaataa
     1141 aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa
     1201 cgtcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc
     1261 gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc
     1321 cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg
     1381 tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta
     1441 ctcctgatga tgcatggtta ctcaccactg cgatccccgg aaaaacagca ttccaggtat
     1501 tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc
     1561 ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg
     1621 ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc
     1681 gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac
     1741 cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga
     1801 aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg
     1861 ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa
     1921 aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt
     1981 ttttctaatc agaattggtt aattggttgt aacactggca gagcattacg ctgacttgac
     2041 gggacggcgc aagctcatga ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag
     2101 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     2161 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     2221 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     2281 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     2341 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     2401 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     2461 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     2521 gtgagcattg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     2581 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     2641 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     2701 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     2761 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     2821 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     2881 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     2941 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     3001 gcaacgcaat taatacgcgt accgctagcc aggaagagtt tgtagaaacg caaaaaggcc
     3061 atccgtcagg atggccttct gcttagtttg atgcctggca gtttatggcg ggcgtcctgc
     3121 ccgccaccct ccgggccgtt gcttcacaac gttcaaatcc gctcccggcg gatttgtcct
     3181 actcaggaga gcgttcaccg acaaacaaca gataaaacga aaggcccagt cttccgactg
     3241 agcctttcgt tttatttgat gcctggcagt tccctactct cgcgttaacg ctagcatgga
     3301 tgttttccca gtcacgacgt tgtaaaacga cggccagtct taagctcggg ccctgcagct
     3361 ctagagctcg aattctacag gtcactaata ccatctaagt agttgattca tagtgactgc
     3421 atatgttgtg ttttacagta ttatgtagtc tgttttttat gcaaaatcta atttaatata
     3481 ttgatattta tatcatttta cgtttctcgt tcaacttt
//
