LOCUS       sequence-418732-        7327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In mammalian cells expresses TP fused to a ZFP36L2 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 7327)
  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 7327)
  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-ZFP36L2-V5H6.MCh.Puro
FEATURES             Location/Qualifiers
     source          1..7327
                     /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             2761..2802
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2812..2829
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(2833..2857)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    2886..3007
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2897..2916)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2951..2970
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     enhancer        3105..3484
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3485..3688
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3639..3659
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3715..3724
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3721..4428
                     /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(3866..3884)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4134..4153
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4429..5028
                     /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(4429..4448)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4925..4945
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5066..5083)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5072..5296
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5321..5338)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5492..6080)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5572..5591)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6251..7042,7043..7111))
                     /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     6874..6893
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7112..7216)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7284..7302
                     /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 cagcatgtcg
     1261 accacacttc tgtccgcctt ctacgatgtc gacttcttgt gcaagacaga gaaatccctg
     1321 gccaacctca acctgaacaa catgctggac aagaaggcgg tggggacgcc tgtggccgcc
     1381 gcccccagct cgggcttcgc gccgggattc ctccgacggc actcggccag caacctgcat
     1441 gcactcgccc accccgcgcc cagccccggc agctgctcgc ccaagttccc gggcgccgct
     1501 aacggcagca gctgcggcag cgcggcggcc ggcggtccga cctcctacgg cacccttaag
     1561 gagccgtcgg ggggcggcgg cacagccctg ctcaacaagg agaacaaatt ccgggaccgc
     1621 tcgtttagcg agaacggcga tcgcagccag cacctcctgc acctgcagca gcagcagaag
     1681 gggggcggcg gctcccagat caactccacg cgctacaaga ccgagctgtg ccggcccttc
     1741 gaggagagcg gcacgtgcaa gtacggcgaa aagtgccagt tcgcgcatgg cttccacgag
     1801 ctgcgcagcc tgactcgcca tccgaagtac aagaccgagc tgtgccgcac ctttcatacc
     1861 atcggcttct gcccctatgg gccgcgctgc cacttcatcc acaacgcgga cgagcggcgg
     1921 cccgcgccgt cggggggcgc ctccggggac ctgcgtgcct ttggcacgcg cgatgcgttg
     1981 cacctgggct tcccgcggga gccgcggccc aagttgcacc acagcctcag cttctcgggc
     2041 ttcccgtcgg gccaccatca gcccccgggc ggcctcgagt cgccgctgct gctcgacagc
     2101 cccacgtcgc gcacgccgcc gccgccctcc tgctcttcgg cctcgtcctg ctcctcctcc
     2161 gcctcctcct gttcctcggc ctccgcggcc tccacgccct cgggcgcccc gacatgctgc
     2221 gcctccgcgg cggccgcggc tgcggccgct ctgctgtacg gcaccggggg cgccgaggac
     2281 ctgctggcgc cgggggcccc gtgcgcggcc tgctcgtcgg cctcgtgcgc caacaacgcc
     2341 ttcgccttcg gtccggagct cagcagcctc atcacgccgc tcgccatcca gacccacaac
     2401 tttgccgccg tggccgccgc cgcctactac cgcagtcagc agcagcagca gcagcagggc
     2461 ctggcgcccc ccgcgcagcc gccggcgccg cccagcgcga ccctccccgc cggggccgcc
     2521 gcacctccct cgccgccctt cagcttccag ctgccgcgcc gcctgtccga ctcgcccgtg
     2581 ttcgacgcgc cccccagccc cccggactcg ctgtcggacc gcgacagcta cctaagcggc
     2641 tccctgagct ccggcagcct cagcggctct gagtctccca gcctcgaccc tggccgccgc
     2701 ctgccaatct tcagccgcct ctccatctcc gacgacaggg atccgggccc gcggttcgaa
     2761 ggtaagccta tccctaaccc tctcctcggt ctcgattcta cgcgtaccgg tcatcatcac
     2821 catcaccatt gaacccagct ttcttgtaca aagtggtgat ggccggccgc ttcgagcaga
     2881 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
     2941 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     3001 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
     3061 ggttttttaa agcaagtaaa acctctacaa atgtggtacg cgttgacatt gattattgac
     3121 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
     3181 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     3241 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
     3301 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     3361 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
     3421 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     3481 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
     3541 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
     3601 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
     3661 acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca ctgcgccacc
     3721 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
     3781 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     3841 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     3901 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     3961 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     4021 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
     4081 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     4141 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     4201 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     4261 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     4321 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     4381 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagat gaccgagtac
     4441 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     4501 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     4561 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     4621 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     4681 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     4741 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     4801 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     4861 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     4921 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     4981 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgact cgagtctaga
     5041 gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     5101 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     5161 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     5221 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     5281 gcggtgggct ctatgggcgg ccgcggcgct cttccgcttc ctcgctcact gactcgctgc
     5341 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5401 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5461 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5521 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5581 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5641 gatacctgtc cgcctttctc tcttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5701 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5761 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5821 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5881 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5941 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6001 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     6061 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6121 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6181 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6241 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6301 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6361 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6421 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6481 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6541 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6601 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6661 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6721 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6781 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6841 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6901 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6961 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     7021 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     7081 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     7141 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     7201 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     7261 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt cggcgcgccg
     7321 cggccgc
//
