LOCUS       sequence-429693-        6065 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant expression of UNK with dual-affinity tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6065)
  AUTHORS   Dominguez D, Freese P, Alexis MS, Su A, Hochman M, Palden T, Bazile 
            C, Lambert NJ, Van Nostrand EL, Pratt GA, Yeo GW, Graveley BR, Burge
            CB
  TITLE     Sequence, Structure, and Context Preferences of Human RNA Binding 
            Proteins.
  JOURNAL   Mol Cell. 2018 Jun 7;70(5):854-867.e9. doi: 
            10.1016/j.molcel.2018.05.001. Epub 2018 Jun 7.
  PUBMED    29883606
REFERENCE   2  (bases 1 to 6065)
  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: 
            "10.1016/j.molcel.2018.05.001"; journalName: "Mol Cell"; date: 
            "2018-06-7- 7"; volume: "70"; issue: "5"; pages: "854-867.e9"
COMMENT     Sequence Label: UNK_6xZNF_pGEX
FEATURES             Location/Qualifiers
     source          1..6065
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     830..849
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(869..946)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(1019..1036)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1190..1778)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1270..1289)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1949..2740,2741..2809))
                     /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"
     primer_bind     2572..2591
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2810..2914)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2982..3000
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     3145..3167
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4218..4331)
                     /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(4341..4364)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(4371..5024)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     complement(4391..4413)
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     protein_bind    5047..5063
                     /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(5071..5077,5078..5093,5094..5099))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             complement(5201..5728)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     5677..5699
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5691..5708
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5692..5708
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5724..5740)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5724..5740)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    5748..5764
                     /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(5772..5778,5779..5796,5797..5802))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(join(5851..6065,1..868))
                     /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"
ORIGIN
        1 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
       61 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
      121 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
      181 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
      241 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
      301 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
      361 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
      421 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
      481 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
      541 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
      601 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
      661 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
      721 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
      781 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
      841 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
      901 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtcggaa tttatgcggt
      961 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct
     1021 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     1081 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     1141 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     1201 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     1261 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     1321 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     1381 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     1441 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     1501 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     1561 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     1621 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     1681 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     1741 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     1801 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     1861 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     1921 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     1981 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     2041 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     2101 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     2161 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     2221 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt
     2281 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     2341 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     2401 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     2461 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     2521 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg gataataccg
     2581 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     2641 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     2701 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     2761 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     2821 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     2881 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     2941 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     3001 cctttcgtct tcaagaatta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     3061 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3121 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3181 caccgaaacg cgcgaggcag atcgtcagtc agtcacgatg cggccgctca gctcacaggc
     3241 accgagtctc cggcggcaga tggcatgtac aggacaggac ccggctgggt ggggctggac
     3301 acagctgagg aaggctgcag gtcgtcactc aggggtggct gttctacgtg ggcaaaggcg
     3361 cagaagggtc ctcggggaca gctgcccgac tgctgcatgt cgttgcactt ggtggacttg
     3421 tagatctcgg ggtggaactg ctgctcggtg cgggtgtggc agtactggca ggcgtctccg
     3481 ttctcacact tgccagggtc tccccactca tccccgtgct tgacgtttgg acatggagac
     3541 gacctgtatt tgtgcttccg ggggctccgc cgccggtcct tgctgttgtg gtagtaggga
     3601 caggcatagc cttggcggca cagccgcggg ggcttcttgc aaggctccgt cttatagttc
     3661 cccagcacat aagcagtctc ttgccaccga ggctcctcgc tgaggatctt ttctatcatg
     3721 gcatggctcg cagccccagc cgactggccc tctatgctcc cctctaccgt ggtctggcca
     3781 ttctgcaagg cctccatggc ctgaagctcc ctgatgtcgt agacagggga gcggaggtca
     3841 tggggcccgt gggcaaaagc gcagtgcagg ccgtttttgg tgcagttgcc tttcgagtct
     3901 gtctcgtgga tgcagattcc agttttgtag taacgaaggt ggtacctgcg ctcagtgtcc
     3961 cctgtggttc tgtgcaggaa tgggcactcg tcgccctccg ggcagaggcc tgtagcctcg
     4021 tcgtacttgg tgcagtagac gtcagggctg taattgaagg tgccgtcccg acggcggatg
     4081 gaccggcggc gccgctggtt cacgaagtgc cagtggaagc aggtgtaggg ccgatgctgc
     4141 gtgcatttgt gttgcacaaa gagtgggcac tgctctgtgc ggaattcttt caggtacgtg
     4201 tagtgctgcg gggatcccgg ttcacgctga ccctgcgggt ggtgttccag acgagcacgc
     4261 agctgttcca gttcaccagc cagaccttca acaacgtgac caccacgcca accggtggtt
     4321 ttttcgtcca ttaatcccag gggcccctgg aacagaactt ccagatccga ttttggagga
     4381 tggtcgccac caccaaacgt ggcttgccag ccctgcaaag gccatgctat atacttgctg
     4441 gatttcaagt acttatcaat ttgtgggata gcttcaatac gttttttaaa acaaactaat
     4501 tttgggaacg catccaggca cattgggtcc atgtataaaa caacatcaag agcgtcatac
     4561 aacatgaagt caggatgggt tacatgatca ccatttaaat atgttttatg acataaacga
     4621 tcttcgaaca ttttcagcat ttcaggtagc ttgctaagaa aatcaacttt gagagtttca
     4681 aagtctttac tatatgcaat tctcgaaaca ccgtatctaa tatccaaaac cgctccttca
     4741 agcattgaaa tctctgcacg ctcttttgga caaccaccca acatgttgtg cttgtcagct
     4801 atataacgta tgatggccat agactgtgtt aatttaacat caccatcaat ataataagga
     4861 agattgggaa actccaaacc caattcaaac tttttgtttc gccatttatc accttcatcg
     4921 cgctcataca aatgctcttc atatttttct tcaagatatt ccaaaagaag tcgagtgggt
     4981 tgcacaaggc ccttaatttt ccaataacct agtatagggg acatgaatac tgtttcctgt
     5041 gtgaaattgt tatccgctca caattccaca cattatacga gccgatgatt aattgtcaac
     5101 agctcatttc agaatatttg ccagaaccgt tatgatgtcg gcgcaaaaaa cattatccag
     5161 aacgggagtg cgccttgagc gacacgaatt atgcagtgat ttacgacctg cacagccata
     5221 ccacagcttc cgatggctgc ctgacgccag aagcattggt gcaccgtgca gtcgataagc
     5281 taattccaac gccatcaaaa ataattcgcg tctggccttc ctgtagccag ctttcatcaa
     5341 cattaaatgt gagcgagtaa caacccgtcg gattctccgt gggaacaaac ggcggattga
     5401 ccgtaatggg ataggtcacg ttggtgtaga tgggcgcatc gtaaccgtgc atctgccagt
     5461 ttgaggggac gacgacagta tcggcctcag gaagatcgca ctccagccag ctttccggca
     5521 ccgcttctgg tgccggaaac caggcaaagc gccattcgcc attcaggctg cgcaactgtt
     5581 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     5641 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
     5701 cggccagtga atccgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     5761 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     5821 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5881 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5941 cgctagcgtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     6001 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     6061 ctgtt
//
