LOCUS       sequence-456256-        6029 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant expression of UNK Dual with dual-affinity tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6029)
  AUTHORS   Harris SE, Hu Y, Bridges K, Cavazos FF Jr, Martyr JG, Guzman BB, 
            Murn J, Aleman MM, Dominguez D
  TITLE     Dissecting RNA selectivity mediated by tandem RNA-binding domains.
  JOURNAL   J Biol Chem. 2025 May;301(5):108435. doi: 10.1016/j.jbc.2025.108435.
            Epub 2025 Mar 20.
  PUBMED    40120682
REFERENCE   2  (bases 1 to 6029)
  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: "J Biol 
            Chem."; date: "2025-05"; volume: "301(5):108435. doi"; pages: " 
            10.1016/j.jbc.2025.108435. Epub 2025 Mar 20"
COMMENT     Sequence Label: UNK_Dual_pGEX
FEATURES             Location/Qualifiers
     source          1..6029
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    38..54
                     /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)."
     primer_bind     62..78
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     62..78
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             74..601
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(94..111)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(94..110)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(103..125)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(703..708,709..724,725..731)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    739..755
                     /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)."
     CDS             778..1431
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1389..1411
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1438..1461
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1471..1584
                     /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"
     primer_bind     complement(2599..2621)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2766..2784)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2852..2956
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2957..3025,3026..3817)
                     /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     complement(3175..3194)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3988..4576
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4477..4496
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4730..4747
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4820..4897
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4898..5980
                     /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     complement(4917..4936)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6029,1..5,6..23,24..30)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
       61 acaggaaaca gctatgacca tgattacgga ttcactggcc gtcgttttac aacgtcgtga
      121 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
      181 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
      241 tggcgaatgg cgctttgcct ggtttccggc accagaagcg gtgccggaaa gctggctgga
      301 gtgcgatctt cctgaggccg atactgtcgt cgtcccctca aactggcaga tgcacggtta
      361 cgatgcgccc atctacacca acgtgaccta tcccattacg gtcaatccgc cgtttgttcc
      421 cacggagaat ccgacgggtt gttactcgct cacatttaat gttgatgaaa gctggctaca
      481 ggaaggccag acgcgaatta tttttgatgg cgttggaatt agcttatcga ctgcacggtg
      541 caccaatgct tctggcgtca ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta
      601 aatcactgca taattcgtgt cgctcaaggc gcactcccgt tctggataat gttttttgcg
      661 ccgacatcat aacggttctg gcaaatattc tgaaatgagc tgttgacaat taatcatcgg
      721 ctcgtataat gtgtggaatt gtgagcggat aacaatttca cacaggaaac agtattcatg
      781 tcccctatac taggttattg gaaaattaag ggccttgtgc aacccactcg acttcttttg
      841 gaatatcttg aagaaaaata tgaagagcat ttgtatgagc gcgatgaagg tgataaatgg
      901 cgaaacaaaa agtttgaatt gggtttggag tttcccaatc ttccttatta tattgatggt
      961 gatgttaaat taacacagtc tatggccatc atacgttata tagctgacaa gcacaacatg
     1021 ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc ttgaaggagc ggttttggat
     1081 attagatacg gtgtttcgag aattgcatat agtaaagact ttgaaactct caaagttgat
     1141 tttcttagca agctacctga aatgctgaaa atgttcgaag atcgtttatg tcataaaaca
     1201 tatttaaatg gtgatcatgt aacccatcct gacttcatgt tgtatgacgc tcttgatgtt
     1261 gttttataca tggacccaat gtgcctggat gcgttcccaa aattagtttg ttttaaaaaa
     1321 cgtattgaag ctatcccaca aattgataag tacttgaaat ccagcaagta tatagcatgg
     1381 cctttgcagg gctggcaagc cacgtttggt ggtggcgacc atcctccaaa atcggatctg
     1441 gaagttctgt tccaggggcc cctgggatta atggacgaaa aaaccaccgg ttggcgtggt
     1501 ggtcacgttg ttgaaggtct ggctggtgaa ctggaacagc tgcgtgctcg tctggaacac
     1561 cacccgcagg gtcagcgtga accgggatcc cctcggtggc aagagactgc ttatgtgctg
     1621 gggaactata agacggagcc ttgcaagaag cccccgcggc tgtgccgcca aggctatgcc
     1681 tgtccctact accacaacag caaggaccgg cggcggagcc cccggaagca caaatacagg
     1741 tcgtctccat gtccaaacgt caagcacggg gatgagtggg gagaccctgg caagtgtgag
     1801 aacggagacg cctgccagta ctgccacacc cgcaccgagc agcagttcca ccccgagatc
     1861 tacaagtcca ccaagtgcaa cgacatgcag cagtcgggca gctgtccccg aggacccttc
     1921 tgcgcctttg cccacgtaga acagccaccc ctgagtgacg acctgcagcc ttcctcagct
     1981 gtgtccggcc agaccacggt agaggggagc atagagggcc agtcggctgg ggctgcgagc
     2041 catgccatga tagaaaagat cctcagcgag gagcctcggt ggcaagagac tgcttatgtg
     2101 ctggggaact ataagacgga gccttgcaag aagcccccgc ggctgtgccg ccaaggctat
     2161 gcctgtccct actaccacaa cagcaaggac cggcggcgga gcccccggaa gcacaaatac
     2221 aggtcgtctc catgtccaaa cgtcaagcac ggggatgagt ggggagaccc tggcaagtgt
     2281 gagaacggag acgcctgcca gtactgccac acccgcaccg agcagcagtt ccaccccgag
     2341 atctacaagt ccaccaagtg caacgacatg cagcagtcgg gcagctgtcc ccgaggaccc
     2401 ttctgcgcct ttgcccacgt agaacagcca cccctgagtg acgacctgca gccttcctca
     2461 gctgtgtcca gccccaccca gccgggtcct gtcctgtaca tgccatctgc cgccggagac
     2521 tcggtgcctg tgagctgagc ggccgcatcg tgactgactg acgatctgcc tcgcgcgttt
     2581 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     2641 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     2701 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtataatt cttgaagacg
     2761 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     2821 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     2881 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     2941 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     3001 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     3061 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     3121 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     3181 tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     3241 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     3301 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     3361 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     3421 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     3481 gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga
     3541 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     3601 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     3661 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     3721 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     3781 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     3841 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     3901 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     3961 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4021 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4081 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     4141 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4201 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4261 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4321 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     4381 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     4441 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     4501 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     4561 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     4621 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     4681 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     4741 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     4801 ttcacaccgc ataaattccg acaccatcga atggcgcaaa acctttcgcg gtatggcatg
     4861 atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga
     4921 tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag
     4981 ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat
     5041 tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac
     5101 ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga
     5161 tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa
     5221 agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct
     5281 ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct
     5341 tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg
     5401 actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc
     5461 attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa
     5521 tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca
     5581 aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca
     5641 gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat
     5701 ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac
     5761 catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc
     5821 tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac
     5881 cacgctagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     5941 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa
     6001 gttagctcac tcattaggca ccccaggct
//
