LOCUS       sequence-456244-        5978 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant expression of UNK with mono-affinity tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5978)
  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 5978)
  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_pGEX_noSBP
FEATURES             Location/Qualifiers
     source          1..5978
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     813..832
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(852..929)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(1002..1019)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1173..1761)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1253..1272)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1932..2723,2724..2792))
                     /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     2555..2574
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2793..2897)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2965..2983
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     3128..3150
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4210..4233)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(4240..4890)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     complement(4260..4282)
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             complement(4891..4908)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    4943..4959
                     /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(4967..4973,4974..4989,4990..4995))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             complement(5097..5624)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     5573..5595
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5587..5604
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5588..5604
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5620..5636)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5620..5636)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    5644..5660
                     /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(5668..5674,5675..5692,5693..5698))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(join(5747..5978,1..851))
                     /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 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
       61 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
      121 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
      181 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
      241 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
      301 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
      361 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
      421 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
      481 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
      541 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
      601 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
      661 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
      721 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
      781 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
      841 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
      901 cgaaaggttt tgcgccattc gatggtgtcg gaatttatgc ggtgtgaaat accgcacaga
      961 tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg
     1021 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     1081 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     1141 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     1201 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     1261 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     1321 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     1381 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     1441 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     1501 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     1561 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     1621 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     1681 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     1741 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     1801 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     1861 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     1921 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     1981 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     2041 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     2101 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     2161 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     2221 agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt
     2281 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     2341 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     2401 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     2461 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     2521 cgaccgagtt gctcttgccc ggcgtcaaca cgggataata ccgcgccaca tagcagaact
     2581 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     2641 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     2701 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     2761 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     2821 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     2881 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt
     2941 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcaagaa
     3001 ttatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     3061 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     3121 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     3181 cagatcgtca gtcagtcacg atgcggccgc ctagctcaca ggcaccgagt ctccggcggc
     3241 agatggcatg tacaggacag gacccggctg ggtggggctg gacacagctg aggaaggctg
     3301 caggtcgtca ctcaggggtg gctgttctac gtgggcaaag gcgcagaagg gtcctcgggg
     3361 acagctgccc gactgctgca tgtcgttgca cttggtggac ttgtagatct cggggtggaa
     3421 ctgctgctcg gtgcgggtgt ggcagtactg gcaggcgtct ccgttctcac acttgccagg
     3481 gtctccccac tcatccccgt gcttgacgtt tggacatgga gacgacctgt atttgtgctt
     3541 ccgggggctc cgccgccggt ccttgctgtt gtggtagtag ggacaggcat agccttggcg
     3601 gcacagccgc gggggcttct tgcaaggctc cgtcttatag ttccccagca cataagcagt
     3661 ctcttgccac cgaggctcct cgctgaggat cttttctatc atggcatggc tcgcagcccc
     3721 agccgactgg ccctctatgc tcccctctac cgtggtctgg ccattctgca aggcctccat
     3781 ggcctgaagc tccctgatgt cgtagacagg ggagcggagg tcatggggcc cgtgggcaaa
     3841 agcgcagtgc aggccgtttt tggtgcagtt gcctttcgag tctgtctcgt ggatgcagat
     3901 tccagttttg tagtaacgaa ggtggtacct gcgctcagtg tcccctgtgg ttctgtgcag
     3961 gaatgggcac tcgtcgccct ccgggcagag gcctgtagcc tcgtcgtact tggtgcagta
     4021 gacgtcaggg ctgtaattga aggtgccgtc ccgacggcgg atggaccggc ggcgccgctg
     4081 gttcacgaag tgccagtgga agcaggtgta gggccgatgc tgcgtgcatt tgtgttgcac
     4141 aaagagtggg cactgctctg tgcggaattc tttcaggtac gtgtagtgct gcggctcgag
     4201 taatcccagg ggcccctgga acagaacttc cagatccgat tttggaggat ggtcgccacc
     4261 accaaacgtg gcttgccagc cctgcaaagg ccatgctata tacttgctgg atttcaagta
     4321 cttatcaatt tgtgggatag cttcaatacg ttttttaaaa caaactaatt ttgggaacgc
     4381 atccaggcac attgggtcca tgtataaaac aacatcaaga gcgtcataca acatgaagtc
     4441 aggatgggtt acatgatcac catttaaata tgttttatga cataaacgat cttcgaacat
     4501 tttcagcatt tcaggtagct tgctaagaaa atcaactttg agagtttcaa agtctttact
     4561 atatgcaatt ctcgaaacac cgtatctaat atccaaaacc gctccttcaa gcattgaaat
     4621 ctctgcacgc tcttttggac aaccacccaa catgttgtgc ttgtcagcta tataacgtat
     4681 gatggccata gactgtgtta atttaacatc accatcaata taataaggaa gattgggaaa
     4741 ctccaaaccc aattcaaact ttttgtttcg ccatttatca ccttcatcgc gctcatacaa
     4801 atgctcttca tatttttctt caagatattc caaaagaagt cgagtgggtt gcacaaggcc
     4861 cttaattttc caataaccta ggatggggga gtgatggtga tggtgatgta taggggacat
     4921 gaatactgtt tcctgtgtga aattgttatc cgctcacaat tccacacatt atacgagccg
     4981 atgattaatt gtcaacagct catttcagaa tatttgccag aaccgttatg atgtcggcgc
     5041 aaaaaacatt atccagaacg ggagtgcgcc ttgagcgaca cgaattatgc agtgatttac
     5101 gacctgcaca gccataccac agcttccgat ggctgcctga cgccagaagc attggtgcac
     5161 cgtgcagtcg ataagctaat tccaacgcca tcaaaaataa ttcgcgtctg gccttcctgt
     5221 agccagcttt catcaacatt aaatgtgagc gagtaacaac ccgtcggatt ctccgtggga
     5281 acaaacggcg gattgaccgt aatgggatag gtcacgttgg tgtagatggg cgcatcgtaa
     5341 ccgtgcatct gccagtttga ggggacgacg acagtatcgg cctcaggaag atcgcactcc
     5401 agccagcttt ccggcaccgc ttctggtgcc ggaaaccagg caaagcgcca ttcgccattc
     5461 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
     5521 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
     5581 cgacgttgta aaacgacggc cagtgaatcc gtaatcatgg tcatagctgt ttcctgtgtg
     5641 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     5701 ctggggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     5761 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5821 cggtttgcgt attgggcgct agcgtggttt ttcttttcac cagtgagacg ggcaacagct
     5881 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     5941 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggc
//
