LOCUS       sequence-418725-        6721 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In mammalian cells expresses TP fused to a CNOT8 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 6721)
  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 6721)
  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-CNOT8-V5H6.MCh.Puro
FEATURES             Location/Qualifiers
     source          1..6721
                     /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             2155..2196
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2206..2223
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(2227..2251)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    2280..2401
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2291..2310)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2345..2364
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     enhancer        2499..2878
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2879..3082
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3033..3053
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3109..3118
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3115..3822
                     /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(3260..3278)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3528..3547
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3823..4422
                     /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(3823..3842)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4319..4339
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4460..4477)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4466..4690
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4715..4732)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4886..5474)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4966..4985)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5645..6436,6437..6505))
                     /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     6268..6287
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6506..6610)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6678..6696
                     /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 cagcatgccc
     1261 gctgccctgg ttgagaattc ccaggtgatc tgcgaagtgt gggcatccaa tcttgaagag
     1321 gagatgcgaa agattcggga aatcgtattg tcctatagtt acatagcgat ggatacagaa
     1381 ttccccggag tcgttgtgcg gcccattggt gaatttcgga gctctatcga ctatcagtat
     1441 cagctcctga ggtgtaatgt ggaccttttg aagatcattc aactgggact gactttcaca
     1501 aacgagaaag gcgaatatcc ctcaggaata aatacctggc aatttaattt caaattcaat
     1561 ctgacagagg atatgtatag ccaagactcc atagatctcc ttgcgaactc tggcctccag
     1621 tttcagaaac acgaagagga aggtatcgac accctgcact ttgccgaact gcttatgacc
     1681 tctggagttg tgctttgtga taacgttaaa tggctgtctt tccacagtgg ttacgatttc
     1741 ggttacatgg taaagctttt gactgattcc cgattgcctg aggaagagca tgagttcttc
     1801 cacattctca atcttttctt tccgtccatt tatgacgtga agtacctgat gaaaagctgc
     1861 aagaacttga aggggggttt gcaggaggta gctgaccaat tggatttgca aagaatcggt
     1921 aggcaacacc aggctggttc cgactccttg cttacgggaa tggcgttctt tagaatgaag
     1981 gaactcttct ttgaggacag cattgatgat gctaagtatt gcggtaggct ttacggtctt
     2041 ggaacaggtg tagcgcagaa gcaaaatgag gatgtagaca gcgctcaaga gaaaatgagt
     2101 attttggcaa taatcaataa catgcagcaa agggatccgg gcccgcggtt cgaaggtaag
     2161 cctatcccta accctctcct cggtctcgat tctacgcgta ccggtcatca tcaccatcac
     2221 cattgaaccc agctttcttg tacaaagtgg tgatggccgg ccgcttcgag cagacatgat
     2281 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     2341 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     2401 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt
     2461 ttaaagcaag taaaacctct acaaatgtgg tacgcgttga cattgattat tgactagtta
     2521 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
     2581 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     2641 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     2701 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     2761 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
     2821 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     2881 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
     2941 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
     3001 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
     3061 ggaggtctat ataagcagag ctctctggct aactagagaa cccactgcgc caccatggtg
     3121 agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac
     3181 atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc
     3241 tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc
     3301 tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc
     3361 gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
     3421 aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag
     3481 ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag
     3541 aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg
     3601 aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc
     3661 aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc
     3721 aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc
     3781 gagggccgcc actccaccgg cggcatggac gagctgtaca agatgaccga gtacaagccc
     3841 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg
     3901 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
     3961 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     4021 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     4081 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     4141 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     4201 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     4261 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc
     4321 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     4381 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gactcgagtc tagagggccc
     4441 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     4501 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     4561 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     4621 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     4681 ggctctatgg gcggccgcgg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4741 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4801 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4861 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4921 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4981 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5041 tgtccgcctt tctctcttcg tgaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5101 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5161 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5221 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5281 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5341 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5401 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5461 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5521 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5581 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5641 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5701 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5761 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5821 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5881 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5941 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6001 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6061 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6121 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6181 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6241 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6301 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6361 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6421 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6481 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6541 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6601 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     6661 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcggcgc gccgcggccg
     6721 c
//
