LOCUS       sequence-418743-        5839 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In mammalian cells expresses TP with a V5H6 tag. Also expresses an 
            mCherry fluorescent protein and puromycin resistance marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5839)
  AUTHORS   Hatfield BM, Weidmann CA, McCutchin CA, Irving PS, Nelson IM, Felder
            S, Weeks KM
  TITLE     Functional Proximity across an mRNA.
  JOURNAL   Biochemistry. 2025 Sep 16;64(18):3854-3865. doi: 
            10.1021/acs.biochem.5c00340. Epub 2025 Aug 12.
  PUBMED    40792674
REFERENCE   2  (bases 1 to 5839)
  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"; doi: 
            "10.1021/acs.biochem.5c00340"; journalName: "Biochemistry"; date: 
            "2025-09-16- 16"; volume: "64"; issue: "18"; pages: "3854-3865"
COMMENT     Sequence Label: pA.CBh.TP-V5H6.MCh.Puro
FEATURES             Location/Qualifiers
     source          1..5839
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..21
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        22..307
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        309..586
                     /label=chicken beta-actin promoter
     intron          587..814
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     protein_bind    819..843
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      844..853
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             853..1239
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2
                     /note="binds to a specific stem-loop structure in the viral
                     RNA (Peabody, 1993)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             1273..1314
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1324..1341
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(1345..1369)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1398..1519
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1409..1428)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1463..1482
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     enhancer        1617..1996
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1997..2200
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2151..2171
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2227..2236
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2233..2940
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2378..2396)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2646..2665
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2941..3540
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2941..2960)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3437..3457
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3578..3595)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3584..3808
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3833..3850)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4004..4592)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4084..4103)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4763..5554,5555..5623))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQSGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5386..5405
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5624..5728)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5796..5814
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 caactttgta tagaaaagtt gcgttacata acttacggta aatggcccgc ctggctgacc
       61 gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg
      121 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      181 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca
      241 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      301 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
      361 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
      421 gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg
      481 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
      541 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
      601 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
      661 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta
      721 gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta
      781 cctggagcac ctgcctgaaa tcactttttt tcaggttggc aagtttgtac aaaaaagcag
      841 gctgccacca tggcttctaa ctttactcag ttcgttctcg tcgacaatgg cggaactggc
      901 gacgtgactg tcgccccaag caacttcgct aacggggtcg ctgaatggat cagctctaac
      961 tcgcgttcac aggcttacaa agtaacctgt agcgttcgtc agagctctgc gcagaatcgc
     1021 aaatacacca tcaaagtcga ggtgcctaaa gtggcaaccc agactgttgg tggtgtagag
     1081 cttcctgtag ccgcatggcg ttcgtactta aatatggaac taaccattcc aattttcgct
     1141 acgaattccg actgcgagct tattgttaag gcaatgcaag gtctcctaaa agatggaaac
     1201 ccgattccct cggccatcgc agcaaactcc ggcatctacg gcggcgctag agatccgggc
     1261 ccgcggttcg aaggtaagcc tatccctaac cctctcctcg gtctcgattc tacgcgtacc
     1321 ggtcatcatc accatcacca ttgaacccag ctttcttgta caaagtggtg atggccggcc
     1381 gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     1441 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     1501 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     1561 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta cgcgttgaca
     1621 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     1681 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     1741 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     1801 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     1861 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     1921 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     1981 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     2041 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     2101 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     2161 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
     2221 cactgcgcca ccatggtgag caagggcgag gaggataaca tggccatcat caaggagttc
     2281 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc
     2341 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt
     2401 ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc
     2461 tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc
     2521 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc
     2581 tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc
     2641 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac
     2701 cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc
     2761 cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc
     2821 gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg
     2881 gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag
     2941 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     3001 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     3061 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     3121 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     3181 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     3241 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     3301 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
     3361 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     3421 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     3481 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga
     3541 ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     3601 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     3661 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     3721 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     3781 catgctgggg atgcggtggg ctctatgggc ggccgcggcg ctcttccgct tcctcgctca
     3841 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3901 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3961 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4021 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4081 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4141 tgccgcttac cggatacctg tccgcctttc tctcttcggg aagcgtggcg ctttctcata
     4201 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4261 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4321 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4381 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4441 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4501 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4561 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4621 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4681 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4741 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4801 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4861 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4921 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4981 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5041 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5101 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5161 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5221 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5281 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5341 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5401 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5461 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc gactgatctt
     5521 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5581 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5641 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5701 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     5761 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     5821 gtcggcgcgc cgcggccgc
//
