LOCUS       sequence-434855-        5480 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant expression of UNK ZnF4-6 with dual-affinity tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5480)
  AUTHORS   Harris SE, Alexis MS, Giri G, Cavazos FF Jr, Hu Y, Murn J, Aleman 
            MM, Burge CB, Dominguez D
  TITLE     Understanding species-specific and conserved RNA-protein 
            interactions in vivo and in vitro.
  JOURNAL   Nat Commun. 2024 Sep 27;15(1):8400. doi: 10.1038/s41467-024-52231-7.
  PUBMED    39333159
REFERENCE   2  (bases 1 to 5480)
  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"; journalName: "Nat 
            Commun."; date: "2024-09-27"; volume: "15(1):8400. doi"; pages: " 
            10.1038/s41467-024-52231-7"
COMMENT     Sequence Label: UNK_ZnF4-6_pGEX
FEATURES             Location/Qualifiers
     source          1..5480
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     378..397
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(616..720)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     788..806
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     951..973
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1439..1552)
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             complement(1562..1585)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(1592..2245)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     complement(1612..1634)
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     protein_bind    2268..2284
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2292..2298,2299..2314,2315..2320))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             complement(2422..2949)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     2898..2920
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2912..2929
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2913..2929
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2945..2961)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2945..2961)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    2969..2985
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2993..2999,3000..3017,3018..3023))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(3072..4154)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4116..4135
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4155..4232)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(4305..4322)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4476..5064)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4556..4575)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5235..5480,1..546,547..615))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
       61 tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
      121 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
      181 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
      241 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
      301 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
      361 cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
      421 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
      481 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
      541 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
      601 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
      661 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
      721 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
      781 ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa gaattataca ctccgctatc
      841 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
      901 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
      961 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagatcg tcagtcagtc
     1021 acgatgcggc cgctcaggac acagctgagg aaggctgcag gtcgtcactc aggggtggct
     1081 gttctacgtg ggcaaaggcg cagaagggtc ctcggggaca gctgcccgac tgctgcatgt
     1141 cgttgcactt ggtggacttg tagatctcgg ggtggaactg ctgctcggtg cgggtgtggc
     1201 agtactggca ggcgtctccg ttctcacact tgccagggtc tccccactca tccccgtgct
     1261 tgacgtttgg acatggagac gacctgtatt tgtgcttccg ggggctccgc cgccggtcct
     1321 tgctgttgtg gtagtaggga caggcatagc cttggcggca cagccgcggg ggcttcttgc
     1381 aaggctccgt cttatagttc cccagcacat aagcagtctc ttgccaccga ggggatcccg
     1441 gttcacgctg accctgcggg tggtgttcca gacgagcacg cagctgttcc agttcaccag
     1501 ccagaccttc aacaacgtga ccaccacgcc aaccggtggt tttttcgtcc attaatccca
     1561 ggggcccctg gaacagaact tccagatccg attttggagg atggtcgcca ccaccaaacg
     1621 tggcttgcca gccctgcaaa ggccatgcta tatacttgct ggatttcaag tacttatcaa
     1681 tttgtgggat agcttcaata cgttttttaa aacaaactaa ttttgggaac gcatccaggc
     1741 acattgggtc catgtataaa acaacatcaa gagcgtcata caacatgaag tcaggatggg
     1801 ttacatgatc accatttaaa tatgttttat gacataaacg atcttcgaac attttcagca
     1861 tttcaggtag cttgctaaga aaatcaactt tgagagtttc aaagtcttta ctatatgcaa
     1921 ttctcgaaac accgtatcta atatccaaaa ccgctccttc aagcattgaa atctctgcac
     1981 gctcttttgg acaaccaccc aacatgttgt gcttgtcagc tatataacgt atgatggcca
     2041 tagactgtgt taatttaaca tcaccatcaa tataataagg aagattggga aactccaaac
     2101 ccaattcaaa ctttttgttt cgccatttat caccttcatc gcgctcatac aaatgctctt
     2161 catatttttc ttcaagatat tccaaaagaa gtcgagtggg ttgcacaagg cccttaattt
     2221 tccaataacc tagtataggg gacatgaata ctgtttcctg tgtgaaattg ttatccgctc
     2281 acaattccac acattatacg agccgatgat taattgtcaa cagctcattt cagaatattt
     2341 gccagaaccg ttatgatgtc ggcgcaaaaa acattatcca gaacgggagt gcgccttgag
     2401 cgacacgaat tatgcagtga tttacgacct gcacagccat accacagctt ccgatggctg
     2461 cctgacgcca gaagcattgg tgcaccgtgc agtcgataag ctaattccaa cgccatcaaa
     2521 aataattcgc gtctggcctt cctgtagcca gctttcatca acattaaatg tgagcgagta
     2581 acaacccgtc ggattctccg tgggaacaaa cggcggattg accgtaatgg gataggtcac
     2641 gttggtgtag atgggcgcat cgtaaccgtg catctgccag tttgagggga cgacgacagt
     2701 atcggcctca ggaagatcgc actccagcca gctttccggc accgcttctg gtgccggaaa
     2761 ccaggcaaag cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     2821 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     2881 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg aatccgtaat
     2941 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     3001 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa cttacattaa
     3061 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     3121 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctagcgt ggtttttctt
     3181 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     3241 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     3301 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca
     3361 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     3421 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     3481 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     3541 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     3601 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     3661 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     3721 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     3781 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     3841 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     3901 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     3961 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     4021 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     4081 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     4141 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     4201 ccgcgaaagg ttttgcgcca ttcgatggtg tcggaattta tgcggtgtga aataccgcac
     4261 agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg
     4321 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4381 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4441 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4501 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4561 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4621 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4681 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4741 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4801 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4861 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     4921 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4981 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5041 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5101 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5161 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5221 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5281 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5341 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5401 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5461 tccgcctcca tccagtctat
//
