LOCUS       sequence-418740-        7105 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In mammalian cells expresses TP fused to a DCP2 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 7105)
  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 7105)
  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-DCP2-V5H6.MCh.Puro
FEATURES             Location/Qualifiers
     source          1..7105
                     /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             2539..2580
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2590..2607
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(2611..2635)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    2664..2785
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2675..2694)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2729..2748
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     enhancer        2883..3262
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3263..3466
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3417..3437
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3493..3502
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3499..4206
                     /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(3644..3662)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3912..3931
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4207..4806
                     /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(4207..4226)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4703..4723
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4844..4861)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4850..5074
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5099..5116)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5270..5858)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5350..5369)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6029..6820,6821..6889))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6652..6671
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6890..6994)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7062..7080
                     /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 cagcatggag
     1261 accaaacggg tggagattcc cggcagcgtc ctggacgatc tctgcagccg atttattttg
     1321 catattccca gcgaggaaag agacaatgca atccgagtgt gttttcagat tgaacttgcc
     1381 cattggtttt acttggattt ctacatgcag aacacaccag gattacctca gtgtgggata
     1441 agagactttg ctaaagctgt cttcagtcat tgtccgtttt tgctgcctca aggtgaagat
     1501 gtggaaaaag ttttggatga atggaaggaa tataaaatgg gagtaccaac atatggtgca
     1561 attattcttg atgagacact tgaaaatgta ctactagttc aggggtacct agcaaaatca
     1621 ggctggggat ttccaaaagg aaaagtaaat aaagaagaag ctcctcatga ttgtgctgct
     1681 agagaggtct ttgaagaaac tggttttgat atcaaagact atatttgtaa ggatgattac
     1741 attgaacttc gaatcaatga ccagcttgct cgtttgtaca tcattccagg aattccaaaa
     1801 gacacaaaat ttaacccaaa aactagaaga gaaattcgga acattgagtg gttctctatt
     1861 gagaaattgc cttgtcatag aaatgatatg acccccaaat ccaaacttgg tttggcacct
     1921 aacaaatttt ttatggccat tccctttatc agaccattaa gggactggct ttctcgaaga
     1981 tttggcgatt cctcagacag tgacaatgga ttttcctcaa ctggtagcac gccggctaaa
     2041 cccactgtgg aaaaattgag tcgaaccaaa ttccgccaca gtcagcagtt atttcctgac
     2101 ggttctcctg gtgaccagtg ggtaaagcac aggcaaccac tgcagcaaaa gccatataat
     2161 aatcattctg aaatgtctga ccttttaaaa ggaaagaatc aaagtatgag gggaaatggc
     2221 agaaaacagt atcaagattc acctaatcaa aagaaaagaa caaatgggct tcagccagca
     2281 aagcagcaga attctttgat gaagtgtgaa aagaaacttc atccacggaa acttcaggat
     2341 aattttgaaa cagatgctgt atatgacttg cctagctcca gtgaagacca gttgctagaa
     2401 catgctgagg gacagcccgt ggcatgtaat ggacattgca agttcccctt ttcatccaga
     2461 gcctttttga gtttcaagtt tgaccataat gctataatga aaatcttgga ccttagggat
     2521 ccgggcccgc ggttcgaagg taagcctatc cctaaccctc tcctcggtct cgattctacg
     2581 cgtaccggtc atcatcacca tcaccattga acccagcttt cttgtacaaa gtggtgatgg
     2641 ccggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag
     2701 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     2761 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     2821 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtacgcg
     2881 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
     2941 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     3001 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     3061 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     3121 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     3181 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     3241 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
     3301 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     3361 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     3421 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag
     3481 agaacccact gcgccaccat ggtgagcaag ggcgaggagg ataacatggc catcatcaag
     3541 gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc
     3601 gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc
     3661 aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc
     3721 aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag
     3781 ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag
     3841 gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc
     3901 ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg
     3961 atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac
     4021 ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg
     4081 cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc
     4141 atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg
     4201 tacaagatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg
     4261 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat
     4321 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg
     4381 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg
     4441 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg
     4501 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc
     4561 aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt
     4621 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc
     4681 ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc
     4741 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt
     4801 gcctgactcg agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct
     4861 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     4921 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     4981 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     5041 agcaggcatg ctggggatgc ggtgggctct atgggcggcc gcggcgctct tccgcttcct
     5101 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     5161 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     5221 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5281 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5341 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     5401 cgaccctgcc gcttaccgga tacctgtccg cctttctctc ttcgggaagc gtggcgcttt
     5461 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     5521 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     5581 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     5641 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     5701 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     5761 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5821 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     5881 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     5941 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6001 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     6061 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     6121 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     6181 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     6241 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     6301 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     6361 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     6421 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     6481 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     6541 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     6601 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     6661 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     6721 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     6781 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     6841 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     6901 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     6961 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     7021 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     7081 cctttcgtcg gcgcgccgcg gccgc
//
