LOCUS       sequence-161232-        3658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3' entry vector containing HA epitope fused to C-terminus and 
            mCherry fused to N-terminus of FRB without pA for rapamycin/rapalog 
            induced dimerization.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3658)
  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 3658)
  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-mCherry-FRB-HA no-pA
FEATURES             Location/Qualifiers
     source          1..3658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(624..747)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             750..1454
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(892..910)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1160..1179
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1354..1377
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             1458..1727
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     CDS             1734..1760
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(1767..1862)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1899..1918)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1900..1918)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1923..1939)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1923..1939)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2041..2060)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2052..2861
                     /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(2119..2138)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3008..3596
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3497..3516
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggccctg cagctctaga gctcgaattc tacaggtcac
      601 taataccatc taagtagttg gttcatagtg actgcatatg ttgtgtttta cagtattatg
      661 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
      721 ctcgttcaac tttcttgtac aaagtggcgg tgagcaaggg cgaggaggac aacatggcca
      781 tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt
      841 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
      901 aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt
      961 acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct
     1021 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg
     1081 tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca
     1141 ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct
     1201 ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc
     1261 tgaaggacgg cggccactac gacgccgagg tcaagaccac ctacaaggcc aagaagcccg
     1321 tgcagctgcc cggcgcctac aacgtcaaca tcaagctgga catcacctcc cacaacgagg
     1381 actacaccat cgtggaacag tacgagcgcg ccgagggccg ccactccacc ggcggcatgg
     1441 acgagctgta caaggtcgag atgtggcatg aaggcctgga agaggcatct cgtttgtact
     1501 ttggggaaag gaacgtgaaa ggcatgtttg aggtgctgga gcccttgcat gctatgatgg
     1561 aacggggccc ccagactctg aaggaaacat cctttaatca ggcctatggt cgagatttaa
     1621 tggaggccca agagtggtgc aggaagtaca tgaaatcagg gaatgtcaag gacctcaccc
     1681 aagcctggga cctctattat catgtgttcc gacgaatctc aaagcaggtc gactatccgt
     1741 acgacgtacc agactacgca gcatagcaac tttattatac aaagttggca ttataaaaaa
     1801 gcattgctta tcaatttgtt gcaacgaaca ggtcactatc agtcaaaata aaatcattat
     1861 ttggagctcc atggtagcgt taacgcggcc gcgatatccc ctatagtgag tcgtattaca
     1921 tggtcatagc tgtttcctgg cagctctggc ccgtgtctca aaatctctga tgttacattg
     1981 cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata
     2041 caaggggtgt tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca
     2101 tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga
     2161 caatctatcg cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag
     2221 gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta
     2281 tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca
     2341 ctgcgatccc cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa
     2401 atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt
     2461 gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg
     2521 gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
     2581 ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt
     2641 tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac
     2701 gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt
     2761 tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga
     2821 ataaattgca gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt
     2881 tgtaacactg gcagagcatt acgctgactt gacgggacgg cgcaagctca tgaccaaaat
     2941 cccttaacgt gagttacgcg tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     3001 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     3061 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     3121 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     3181 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     3241 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     3301 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     3361 gaacgaccta caccgaactg agatacctac agcgtgagca ttgagaaagc gccacgcttc
     3421 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     3481 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     3541 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     3601 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgtt
//
