LOCUS       sequence-418715-        7516 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In mammalian cells expresses TP fused to a 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 7516)
  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 7516)
  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-CNOT6-V5H6.MCh.Puro
FEATURES             Location/Qualifiers
     source          1..7516
                     /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             2950..2991
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3001..3018
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(3022..3046)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    3075..3196
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3086..3105)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3140..3159
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     enhancer        3294..3673
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3674..3877
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3828..3848
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3904..3913
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3910..4617
                     /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(4055..4073)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4323..4342
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4618..5217
                     /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(4618..4637)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5114..5134
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5255..5272)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5261..5485
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5510..5527)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5681..6269)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5761..5780)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6440..7231,7232..7300))
                     /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     7063..7082
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7301..7405)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7473..7491
                     /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 cagcatgcct
     1261 aaagagaagt atgagcctcc tgacccacga aggatgtata cgataatgag ttctgaagaa
     1321 gcagctaatg gcaaaaaatc ccactgggcc gaattggaaa ttagcggcaa agtcaggagt
     1381 ctctctgctt cactgtggag cctcactcac ctgacagcgc tccatctcag tgacaattcc
     1441 ctgagcagga ttccctcaga catcgcaaaa cttcataacc ttgtttacct tgatcttagt
     1501 tcaaataaga tccgatccct tcctgcggaa ctcggtaaca tggtaagcct gcgcgagttg
     1561 caccttaata acaacttgct ccgagttctc ccttttgagc tggggaagct tttccaactt
     1621 caaaccctcg gtctcaaagg caatccgttg acccaggaca tcttgaatct gtaccaagag
     1681 cccgatggta cacggcggct tctcaattat ctcctggaca atctttccgg gacggcaaag
     1741 aggattacca ccgagcagcc tccacctcgc tcctggatca tgttgcaaga gccggaccga
     1801 acccgaccta cagctctgtt cagtgtcatg tgctacaatg ttctttgtga taaatacgct
     1861 accagacaac tttacggtta ttgcccgagt tgggcactca attgggacta ccgcaaaaag
     1921 gcgattatcc aggagatctt gtcatgtaat gccgatattg tgtcactgca ggaggtggag
     1981 acagaacaat actactcttt ttttttggtc gaactgaaag agaggggata taatgggttc
     2041 ttcagcccga agagtcgggc caggaccatg agcgaacaag agcgaaagca tgtagatggt
     2101 tgcgcgatct tcttcaaaac tgaaaaattc acattggtac aaaagcacac ggtagaattt
     2161 aatcagttgg cgatggccaa ttctgagggc tcagaagcaa tgcttaacag agttatgacc
     2221 aaagacaata taggagttgc agttctcttg gagcttagga aggagagtat cgagatgccc
     2281 agcggcaaac cacacctcgg caccgagaaa cagctgatcc tcgttgccaa tgcgcacatg
     2341 cactgggacc ctgaatattc agacgtcaaa ctggtacaaa ctatgatgtt tctgtccgaa
     2401 gttaaaaaca ttattgacaa agcgtcaaga aatctcaaaa gcagtgtgct tggcgaattt
     2461 gggacgatac ccttggtcct gtgtgctgat ctcaattctc tccccgattc aggtgtagtt
     2521 gagtatttgt ccacgggtgg tgtagaaacg aatcataagg atttcaaaga actgcgctac
     2581 aacgagtctc ttacgaactt ttcttgccac ggcaagaacg gtacaacgaa cggaaggata
     2641 actcacggtt tcaaattgca atcagcctac gagtctggct tgatgcccta cactaactat
     2701 accttcgatt ttaaaggtat cattgattac attttctata gtaaaccaca gctgaataca
     2761 ttgggcatct tgggtccact ggatcaccac tggctcgtgg agaataacat aagcgggtgc
     2821 cctcaccccc ttataccatc agaccatttc tcactgtttg cccagcttga gctgttgctg
     2881 ccattcctgc ctcaagtgaa cgggatacat cttcccggac gaagaaggga tccgggcccg
     2941 cggttcgaag gtaagcctat ccctaaccct ctcctcggtc tcgattctac gcgtaccggt
     3001 catcatcacc atcaccattg aacccagctt tcttgtacaa agtggtgatg gccggccgct
     3061 tcgagcagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     3121 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     3181 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     3241 ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtacgc gttgacattg
     3301 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
     3361 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     3421 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     3481 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     3541 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     3601 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     3661 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
     3721 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     3781 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
     3841 taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac
     3901 tgcgccacca tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg
     3961 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag
     4021 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
     4081 cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac
     4141 gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag
     4201 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc
     4261 ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac
     4321 ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc
     4381 gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac
     4441 tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc
     4501 tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa
     4561 cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagatg
     4621 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     4681 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     4741 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     4801 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     4861 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     4921 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     4981 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     5041 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     5101 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     5161 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgactc
     5221 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     5281 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     5341 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     5401 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     5461 gctggggatg cggtgggctc tatgggcggc cgcggcgctc ttccgcttcc tcgctcactg
     5521 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5581 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5641 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5701 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5761 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5821 cgcttaccgg atacctgtcc gcctttctct cttcgggaag cgtggcgctt tctcatagct
     5881 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5941 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6001 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6061 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6121 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6181 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6241 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6301 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6361 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6421 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6481 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6541 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6601 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6661 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6721 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6781 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6841 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6901 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6961 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     7021 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     7081 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7141 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     7201 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     7261 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     7321 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7381 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     7441 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     7501 ggcgcgccgc ggccgc
//
