LOCUS       sequence-289959-        6467 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of HsTNRC6A_1209-1709-M2WA-CIM1-2mut-F1359A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6467)
  AUTHORS   Huntzinger E, Kuzuoglu-Ozturk D, Braun JE, Eulalio A, Wohlbold L, 
            Izaurralde E
  TITLE     The interactions of GW182 proteins with PABP and deadenylases are 
            required for both translational repression and degradation of miRNA 
            targets.
  JOURNAL   Nucleic Acids Res. 2013 Jan;41(2):978-94. doi: 10.1093/nar/gks1078. 
            Epub 2012 Nov 21.
  PUBMED    23172285
REFERENCE   2  (bases 1 to 6467)
  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: 
            "10.1093/nar/gks1078"; journalName: "Nucleic Acids Res"; date: 
            "2013-01"; volume: "41"; issue: "2"; pages: "978-94"
COMMENT     Sequence Label: pGEX-6P-1-HsTNRC6A_1209-1709-M2WA-CIM1-2mut-F1...
FEATURES             Location/Qualifiers
     source          1..6467
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..941
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    1541..1552
                     /label=ZF binding site
                     /bound_moiety="CCR5TC zinc-finger domain"
                     /note="target sequence for the CCR5TC zinc-finger domain 
                     from the CCR5-224 (+) zinc-finger nuclease (Perez et al., 
                     2008; Gross et al., 2013)"
     primer_bind     complement(2517..2539)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2684..2702)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2770..2874
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2875..2943,2944..3735)
                     /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(3093..3112)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3906..4494
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4395..4414
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4648..4665
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4738..4815
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             4816..5898
                     /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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4835..4854)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5911..5932
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5947..5952,5953..5970,5971..5977)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5985..6001
                     /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     6009..6025
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6009..6025
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6021..6467,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6041..6058)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6041..6057)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6050..6072)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gaagttctgt tccaggggcc cctgggatcc ccaaacctgt
      961 tggtgaagca gcagactcca ccatctcagc agcagccact ccatcagcca gccatgaagt
     1021 ctttccttga caatgtcatg ccccacacta cacctgagct gcaaaaaggg ccatcaccaa
     1081 taaatgcttt cagcaacttc cctataggct tgaactcaaa cttgaatgta aatatggata
     1141 tgaacagtat taaagagcca gctgctgctg ctagggctgc tacgacagtg gacagcattt
     1201 ctgtgaacac atctttggat caaaactcca gcaaacatgg tgctatttca agtggtttca
     1261 ggctggaaga gtctccattt gttccctatg actttatgaa cagcagtact tcaccagcca
     1321 gtcctccagg ttcaatagga gatggctggc cacgtgccaa atcgcctaac ggctctagca
     1381 gtgttaattg gccaccagaa gctcgtcctg gtgagccatg gaaaggttat ccaaacattg
     1441 accctgaaac tgacccttac gtcactcctg gcagtgtcat aaacaatctt tcaattaata
     1501 ctgtgcggga agttgaccac ctcagggaca ggaacagtgg gtcatcctca tccttgacaa
     1561 ccacgctgcc ttcaactagt gccgcgtcat ccattcgtgc ctccaactac aacgttcccc
     1621 tcagcagtac agcacaaagc acttcagcca gaaatagtga ttccaaattg acagcgtctc
     1681 ctggttcagt tacaaacacc tctctggctc atgagctggc gaaggtccct ttgccaccta
     1741 aaaacatcac tgctccgtcc cgcccacctc cgggactgac tggtcagaag ccacccttgt
     1801 ctacggcgga taattctccc cttcgtatag gtggaggagc gggaaattct gacgccagat
     1861 ataccccagg ttccagcgcg ggtgagagca gctcagggag aataacaaat tggcttgttc
     1921 taaaaaacct tacacctcag atcgatggct caactctgcg cactctgtgc atgcagcacg
     1981 gcccgctgat cacattccac ctgaacctcc ctcacggaaa tgctctggtc cgctacagtt
     2041 caaaagaaga ggtagtggag gcacaaaagt ctctgcacat gtgtgtactg gggaacacta
     2101 ctattcttgc tgagtttgcc agtgaagagg agatcagtcg tttctttgca caaagccagt
     2161 ctctgacccc ttctcccggc tggcagtctc tcgggtccag ccagagccgg ctgggctccc
     2221 tcgactgttc ccactcattc tccagccgga ccgatctcaa tcactggaat ggtgctgggc
     2281 tgtcgggaac taactgtgga gaccttcacg gcacttcagc tgctgctacc ccgcattatt
     2341 ccacaagcgc tgctgctccc ccaagcagca gcgacccccg aggaattagc agcccatctc
     2401 ccattaacgc ttttctttct gttgaccacc tgggtggggg tggagagtcc atgtaagcgg
     2461 ccgcatcgtg actgactgac gatctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc
     2521 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     2581 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag
     2641 tcacgtagcg atagcggagt gtataattct tgaagacgaa agggcctcgt gatacgccta
     2701 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     2761 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2821 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     2881 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     2941 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     3001 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     3061 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
     3121 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     3181 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     3241 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     3301 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     3361 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca
     3421 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     3481 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     3541 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     3601 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     3661 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     3721 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     3781 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     3841 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3901 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3961 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4021 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     4081 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     4141 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4201 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4261 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4321 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4381 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4441 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4501 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     4561 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     4621 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     4681 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat aaattccgac
     4741 accatcgaat ggtgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa
     4801 ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc
     4861 tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg
     4921 gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa
     4981 ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg
     5041 ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg
     5101 gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc
     5161 gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct
     5221 gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc
     5281 atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc
     5341 gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt
     5401 ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa
     5461 cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag
     5521 ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc
     5581 gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat
     5641 accgaagaca gctcatgtta tatcccgccg tcaaccacca tcaaacagga ttttcgcctg
     5701 ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc
     5761 aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa
     5821 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     5881 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     5941 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     6001 atttcacaca ggaaacagct atgaccatga ttacggattc actggccgtc gttttacaac
     6061 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     6121 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     6181 gcctgaatgg cgaatggcgc tttgcctggt ttccggcacc agaagcggtg ccggaaagct
     6241 ggctggagtg cgatcttcct gaggccgata ctgtcgtcgt cccctcaaac tggcagatgc
     6301 acggttacga tgcgcccatc tacaccaacg taacctatcc cattacggtc aatccgccgt
     6361 ttgttcccac ggagaatccg acgggttgtt actcgctcac atttaatgtt gatgaaagct
     6421 ggctacagga aggccagacg cgaattattt ttgatggcgt tggaatt
//
