LOCUS       sequence-418712-        6699 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In mammalian cells expresses TP fused to a CNOT7 that does not 
            interact with CNOT6 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 6699)
  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 6699)
  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-CNOT7(minusCNOT6interaction)-V5H6.MC...
FEATURES             Location/Qualifiers
     source          1..6699
                     /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..585
                     /label=chicken beta-actin promoter
     intron          586..813
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     protein_bind    818..842
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      843..852
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             852..1238
                     /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             2133..2174
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2184..2201
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(2205..2229)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    2258..2379
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2269..2288)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2323..2342
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     enhancer        2477..2856
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2857..3060
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3011..3031
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3087..3096
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3093..3800
                     /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(3238..3256)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3506..3525
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3801..4400
                     /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(3801..3820)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4297..4317
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4438..4455)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4444..4668
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4693..4710)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4864..5452)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4944..4963)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5623..6414,6415..6483))
                     /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     6246..6265
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6484..6588)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6656..6674
                     /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 gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg
      481 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg
      541 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc
      601 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
      661 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
      721 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac
      781 ctggagcacc tgcctgaaat cacttttttt caggttggca agtttgtaca aaaaagcagg
      841 ctgccaccat ggcttctaac tttactcagt tcgttctcgt cgacaatggc ggaactggcg
      901 acgtgactgt cgccccaagc aacttcgcta acggggtcgc tgaatggatc agctctaact
      961 cgcgttcaca ggcttacaaa gtaacctgta gcgttcgtca gagctctgcg cagaatcgca
     1021 aatacaccat caaagtcgag gtgcctaaag tggcaaccca gactgttggt ggtgtagagc
     1081 ttcctgtagc cgcatggcgt tcgtacttaa atatggaact aaccattcca attttcgcta
     1141 cgaattccga ctgcgagctt attgttaagg caatgcaagg tctcctaaaa gatggaaacc
     1201 cgattccctc ggccatcgca gcaaactccg gcatctacgg cggcgctagc agcatgccag
     1261 cggcaactgt agatcatagc caaagaattt gtgaagtttg ggcttgcaac ttggatgaag
     1321 agatgaagaa aattcgtcaa gttatccgaa aatataatta cgttgctatg gacaccgagt
     1381 ttccaggtgt ggttgcaaga cccattggag aattcaggag caatgctgac tatcaatacc
     1441 aactattgcg gtggaatgta gactggttaa agataattca gctaggactg acatttatga
     1501 atgagcaagg agaataccct ccaggaactt caacttggca gtttaatttt aaatttaatt
     1561 tgacggagga catgtatgcc caggactcta tagagctact aacaacatct ggtatccagt
     1621 ttaaaaaaca tgaggaggaa ggaattgaaa cccagtactt tgcagaactt cttatgactt
     1681 ctggagtggt cctctgtgaa ggggtcaaat ggttgtcatt tcatagcggt tacgactttg
     1741 gctacttaat caaaatccta accaactcta acttgcctga agaagaactt gacttctttg
     1801 agatccttcg attgtttttt cctgtcattt atgatgtgaa gtacctcatg aagagctgca
     1861 aaaatctcaa aggtggatta caggaggtgg cagaacagtt agagctggaa cggataggac
     1921 cacaacatca ggcaggatct gattcattgc tcacaggaat ggcctttttc aaaatgagag
     1981 aaatgttctt tgaagatcat attgatgatg ccaaatattg tggtcatttg tatggccttg
     2041 gttctggttc atcctatgta cagaatggca cagggaatgc atatgaagag gaagccaaca
     2101 agcagtcaag ggatccgggc ccgcggttcg aaggtaagcc tatccctaac cctctcctcg
     2161 gtctcgattc tacgcgtacc ggtcatcatc accatcacca ttgaacccag ctttcttgta
     2221 caaagtggtg atggccggcc gcttcgagca gacatgataa gatacattga tgagtttgga
     2281 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     2341 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     2401 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac
     2461 aaatgtggta cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
     2521 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     2581 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     2641 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     2701 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     2761 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     2821 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
     2881 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
     2941 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
     3001 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
     3061 ctctggctaa ctagagaacc cactgcgcca ccatggtgag caagggcgag gaggataaca
     3121 tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc
     3181 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
     3241 agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt
     3301 tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc
     3361 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
     3421 tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc
     3481 gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     3541 cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc
     3601 tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga
     3661 agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca
     3721 acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg
     3781 gcatggacga gctgtacaag atgaccgagt acaagcccac ggtgcgcctc gccacccgcg
     3841 acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc
     3901 gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc
     3961 tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg
     4021 cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc
     4081 gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg
     4141 cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc
     4201 accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg
     4261 ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc
     4321 tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga
     4381 cccgcaagcc cggtgcctga ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct
     4441 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     4501 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     4561 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     4621 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatgggc ggccgcggcg
     4681 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4741 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4801 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4861 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4921 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4981 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tctcttcggg
     5041 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5101 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5161 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5221 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5281 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5341 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5401 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5461 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5521 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5581 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5641 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5701 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5761 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5821 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5881 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5941 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6001 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6061 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6121 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6181 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6241 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6301 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6361 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6421 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6481 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6541 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6601 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     6661 gcgtatcacg aggccctttc gtcggcgcgc cgcggccgc
//
