LOCUS       sequence-418727-        7510 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In mammalian cells expresses TP fused to a CNOT6L 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 7510)
  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 7510)
  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-CNOT6L-V5H6.MCh.Puro
FEATURES             Location/Qualifiers
     source          1..7510
                     /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             2944..2985
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2995..3012
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(3016..3040)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    3069..3190
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3080..3099)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3134..3153
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     enhancer        3288..3667
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3668..3871
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3822..3842
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3898..3907
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3904..4611
                     /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(4049..4067)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4317..4336
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4612..5211
                     /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(4612..4631)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5108..5128
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5249..5266)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5255..5479
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5504..5521)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5675..6263)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5755..5774)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6434..7225,7226..7294))
                     /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     7057..7076
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7295..7399)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7467..7485
                     /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 cagcatgcga
     1261 ctgataggta tgccaaaaga aaaatacgat cctccggacc cgagaaggat ttatacgatc
     1321 atgagtgctg aggaggtggc gaatggcaaa aagtctcatt gggccgagtt ggagatatcc
     1381 ggacgggtca gaagtctttc tacgtcactg tggtcactta cgcatctcac tgcacttcac
     1441 cttaatgata actacctttc ccgaattccg ccagacatcg caaaacttca taacctcgtt
     1501 tatcttgacc tgagttccaa taaattgcga tctctgcccg ctgagcttgg taacatggtg
     1561 tctttgagag aactcctttt gaacaacaat cttctcagag tcttgccgta cgagctcggc
     1621 aggttgttcc aactccaaac gttgggcctt aaaggaaatc ctctgagcca agatatactg
     1681 aatctgtatc aagaccccga tggtacgcgg aaattgttga attttatgct cgacaacttg
     1741 gcggtgcacc ccgagcagtt gccaccccgg ccctggatta cactcaagga acgcgatcag
     1801 attctgccgt ctgcttcctt taccgttatg tgttacaacg tgctgtgcga taagtatgca
     1861 acaaggcaac tgtatggcta ttgcccctca tgggcattga attgggaata ccgaaaaaaa
     1921 gggattatgg aagaaattgt gaactgcgac gccgatatca tctcactcca ggaagtagag
     1981 acagagcagt atttcacctt gttcctccca gccctgaaag aacgaggtta cgatggattt
     2041 ttctcaccca agtctagggc aaaaatcatg tccgaacaag aaaggaagca cgtggatggg
     2101 tgtgcaatct ttttcaagac ggagaagttt accttggtgc aaaagcatac cgtggagttt
     2161 aatcaggtcg caatggccaa cagcgatgga tctgaagcga tgctgaaccg cgttatgaca
     2221 aaagacaaca ttggagttgc agtagtgttg gaggtccata aagaattgtt cggagctggt
     2281 atgaaaccaa tacacgccgc agacaaacaa ctgttgatcg tagctaacgc ccatatgcat
     2341 tgggacccgg aatacagcga cgtgaaactc atccagacca tgatgtttgt ttccgaagtg
     2401 aaaaacatcc tcgaaaaagc tagttcccgc ccaggcagcc caacagcaga tccaaattcc
     2461 atcccactcg tcttgtgtgc tgatctgaat tcattgccgg acagtggcgt ggttgagtat
     2521 cttagcaatg gcggtgtggc cgacaatcac aaagatttta aagagctcag atataatgaa
     2581 tgcctcatga atttcagctg caacggtaag aacggctcca gtgagggtcg catcactcat
     2641 gggtttcaac tgaaatcagc gtacgaaaat aatctcatgc cgtacacaaa ctacaccttt
     2701 gatttcaaag gtgttattga ttatatcttc tatagcaaga cccacatgaa tgttctggga
     2761 gtgctgggcc ctctggaccc acagtggctg gtcgaaaata atataacagg ctgcccacac
     2821 ccgcatatac caagtgatca cttctcactt ttgacgcagc ttgaacttca ccctccattg
     2881 cttcctctcg tgaatggcgt gcacctccca aaccggagaa gggatccggg cccgcggttc
     2941 gaaggtaagc ctatccctaa ccctctcctc ggtctcgatt ctacgcgtac cggtcatcat
     3001 caccatcacc attgaaccca gctttcttgt acaaagtggt gatggccggc cgcttcgagc
     3061 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     3121 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     3181 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     3241 ggaggttttt taaagcaagt aaaacctcta caaatgtggt acgcgttgac attgattatt
     3301 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     3361 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     3421 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     3481 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     3541 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     3601 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     3661 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
     3721 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     3781 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
     3841 tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac ccactgcgcc
     3901 accatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt catgcgcttc
     3961 aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag
     4021 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg
     4081 cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag
     4141 caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag
     4201 cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag
     4261 gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc
     4321 gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta ccccgaggac
     4381 ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg ccactacgac
     4441 gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacaac
     4501 gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt ggaacagtac
     4561 gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa gatgaccgag
     4621 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     4681 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     4741 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     4801 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     4861 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     4921 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     4981 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     5041 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     5101 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     5161 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg actcgagtct
     5221 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     5281 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     5341 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     5401 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     5461 gatgcggtgg gctctatggg cggccgcggc gctcttccgc ttcctcgctc actgactcgc
     5521 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5581 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5641 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5701 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5761 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5821 ccggatacct gtccgccttt ctctcttcgg gaagcgtggc gctttctcat agctcacgct
     5881 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5941 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6001 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6061 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6121 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6181 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6241 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6301 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6361 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6421 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6481 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6541 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6601 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6661 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6721 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6781 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6841 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6901 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6961 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     7021 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     7081 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7141 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7201 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7261 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7321 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7381 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     7441 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcggcgcg
     7501 ccgcggccgc
//
