LOCUS       sequence-418737-        6700 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In mammalian cells expresses TP fused to an inactive CNOT7 that does
            not interact with NOT1 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 6700)
  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 6700)
  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-(inactive)CNOT7(minusNOT1interaction)-V5H6.MCh.Puro
FEATURES             Location/Qualifiers
     source          1..6700
                     /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             2134..2175
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2185..2202
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(2206..2230)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    2259..2380
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2270..2289)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2324..2343
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     enhancer        2478..2857
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2858..3061
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3012..3032
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3088..3097
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3094..3801
                     /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(3239..3257)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3507..3526
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3802..4401
                     /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(3802..3821)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4298..4318
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4439..4456)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4445..4669
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4694..4711)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4865..5453)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4945..4964)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5624..6415,6416..6484))
                     /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     6247..6266
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6485..6589)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6657..6675
                     /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 cagcatgcca
     1261 gcggcaactg tagatcatag ccaaagaatt tgtgaagttt gggcttgcaa cttggatgaa
     1321 gagatgaaga aaattcgtca agttatccga aaatataatt acgttgctat ggccaccgca
     1381 tttccaggtg tggttgcaag acccattgga gaattcagga gcaatgctga ctatcaatac
     1441 caactattgc ggtgtaatgt agacttgtta aagataattc agctaggact gacatttatg
     1501 aatgagcaag gagaataccc tccaggaact tcaacttggc agtttaattt taaatttaat
     1561 ttgacggagg acatgtatgc ccaggactct atagagctac taacaacatc tggtatccag
     1621 tttaaaaaac atgaggagga aggaattgaa acccagtact ttgcagaact tcttcggact
     1681 tctggagtgg tcctctgtga aggggtcaaa tggttgtcat ttcatagcgg ttacgacttt
     1741 ggctacttaa tcaaaatcct aaccaactct aacttgcctg aagaagaact tgacttcttt
     1801 gagatccttc gattgttttt tcctgtcatt tatgatgtga agtacctcat gaagagctgc
     1861 aaaaatctca aaggtggatt acaggaggtg gcagaacagt tagagctgga acggatagga
     1921 ccacaagctc aggcaggatc tgcttcattg ctcacaggaa tggccttttt caaaatgaga
     1981 gaaatgttct ttgaagatca tattgatgat gccaaatatt gtggtcattt gtatggcctt
     2041 ggttctggtt catcctatgt acagaatggc acagggaatg catatgaaga ggaagccaac
     2101 aagcagtcaa gggatccggg cccgcggttc gaaggtaagc ctatccctaa ccctctcctc
     2161 ggtctcgatt ctacgcgtac cggtcatcat caccatcacc attgaaccca gctttcttgt
     2221 acaaagtggt gatggccggc cgcttcgagc agacatgata agatacattg atgagtttgg
     2281 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     2341 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     2401 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta
     2461 caaatgtggt acgcgttgac attgattatt gactagttat taatagtaat caattacggg
     2521 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     2581 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     2641 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     2701 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     2761 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
     2821 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
     2881 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
     2941 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
     3001 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
     3061 tctctggcta actagagaac ccactgcgcc accatggtga gcaagggcga ggaggataac
     3121 atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc
     3181 cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc
     3241 aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag
     3301 ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag
     3361 ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg
     3421 gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg
     3481 cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag
     3541 gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg
     3601 ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag
     3661 aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac
     3721 aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc
     3781 ggcatggacg agctgtacaa gatgaccgag tacaagccca cggtgcgcct cgccacccgc
     3841 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     3901 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     3961 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
     4021 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     4081 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
     4141 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac
     4201 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
     4261 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg
     4321 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     4381 acccgcaagc ccggtgcctg actcgagtct agagggcccg tttaaacccg ctgatcagcc
     4441 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     4501 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     4561 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     4621 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggg cggccgcggc
     4681 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4741 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4801 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4861 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4921 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4981 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctctcttcgg
     5041 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5101 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5161 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5221 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5281 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5341 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5401 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5461 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5521 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5581 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5641 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5701 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5761 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5821 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5881 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5941 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6001 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6061 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6121 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6181 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6241 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6301 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6361 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6421 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6481 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6541 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6601 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     6661 ggcgtatcac gaggcccttt cgtcggcgcg ccgcggccgc
//
