LOCUS       sequence-289969-        6470 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of HsTNRC6A_1209-1709-CIM1-2mut-F1359A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6470)
  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 6470)
  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-CIM1-2mut-F1359A_S
FEATURES             Location/Qualifiers
     source          1..6470
                     /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    1544..1555
                     /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(2520..2542)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2687..2705)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2773..2877
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2878..2946,2947..3738)
                     /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(3096..3115)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3909..4497
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4398..4417
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4651..4668
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4741..4818
                     /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             4819..5901
                     /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(4838..4857)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5914..5935
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5950..5955,5956..5973,5974..5980)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5988..6004
                     /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     6012..6028
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6012..6028
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6024..6470,1..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(6044..6061)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6044..6060)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6053..6075)
                     /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 ttggacgaca gtggacagca
     1201 tttctgtgaa cacatctttg gatcaaaact ccagcaaaca tggtgctatt tcaagtggtt
     1261 tcaggctgga agagtctcca tttgttccct atgactttat gaacagcagt acttcaccag
     1321 ccagtcctcc aggttcaata ggagatggct ggccacgtgc caaatcgcct aacggctcta
     1381 gcagtgttaa ttggccacca gaagctcgtc ctggtgagcc atggaaaggt tatccaaaca
     1441 ttgaccctga aactgaccct tacgtcactc ctggcagtgt cataaacaat ctttcaatta
     1501 atactgtgcg ggaagttgac cacctcaggg acaggaacag tgggtcatcc tcatccttga
     1561 acaccacgct gccttcaact agtgcctggt catccattcg tgcctccaac tacaacgttc
     1621 ccctcagcag tacagcacaa agcacttcag ccagaaatag tgattccaaa ttgacatggt
     1681 ctcctggttc agttacaaac acctctctgg ctcatgagct gtggaaggtc cctttgccac
     1741 ctaaaaacat cactgctccg tcccgcccac ctccgggact gactggtcag aagccaccct
     1801 tgtctacgtg ggataattct ccccttcgta taggtggagg atggggaaat tctgacgcca
     1861 gatatacccc aggttccagc tggggtgaga gcagctcagg gagaataaca aattggcttg
     1921 ttctaaaaaa ccttacacct cagatcgatg gctcaactct gcgcactctg tgcatgcagc
     1981 acggcccgct gatcacattc cacctgaacc tccctcacgg aaatgctctg gtccgctaca
     2041 gttcaaaaga agaggtagtg gaggcacaaa agtctctgca catgtgtgta ctggggaaca
     2101 ctactattct tgctgagttt gccagtgaag aggagatcag tcgtttcttt gcacaaagcc
     2161 agtctctgac cccttctccc ggctggcagt ctctcgggtc cagccagagc cggctgggct
     2221 ccctcgactg ttcccactca ttctccagcc ggaccgatct caatcactgg aatggtgctg
     2281 ggctgtcggg aactaactgt ggagaccttc acggcacttc agctgctgct accccgcatt
     2341 attccacaag cgctgctgct cccccaagca gcagcgaccc ccgaggaatt agcagcccat
     2401 ctcccattaa cgcttttctt tctgttgacc acctgggtgg gggtggagag tccatgtaag
     2461 cggccgcatc gtgactgact gacgatctgc ctcgcgcgtt tcggtgatga cggtgaaaac
     2521 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     2581 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc
     2641 cagtcacgta gcgatagcgg agtgtataat tcttgaagac gaaagggcct cgtgatacgc
     2701 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     2761 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2821 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     2881 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     2941 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     3001 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     3061 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     3121 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     3181 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     3241 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     3301 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     3361 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     3421 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     3481 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     3541 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     3601 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     3661 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     3721 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     3781 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     3841 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3901 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3961 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4021 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     4081 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4141 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4201 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4261 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4321 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4381 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4441 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4501 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4561 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4621 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4681 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg cataaattcc
     4741 gacaccatcg aatggtgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
     4801 caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt
     4861 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
     4921 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
     4981 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
     5041 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
     5101 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
     5161 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
     5221 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
     5281 cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg
     5341 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
     5401 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
     5461 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
     5521 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
     5581 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac
     5641 gataccgaag acagctcatg ttatatcccg ccgtcaacca ccatcaaaca ggattttcgc
     5701 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     5761 ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
     5821 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     5881 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
     5941 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
     6001 acaatttcac acaggaaaca gctatgacca tgattacgga ttcactggcc gtcgttttac
     6061 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     6121 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     6181 gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc accagaagcg gtgccggaaa
     6241 gctggctgga gtgcgatctt cctgaggccg atactgtcgt cgtcccctca aactggcaga
     6301 tgcacggtta cgatgcgccc atctacacca acgtaaccta tcccattacg gtcaatccgc
     6361 cgtttgttcc cacggagaat ccgacgggtt gttactcgct cacatttaat gttgatgaaa
     6421 gctggctaca ggaaggccag acgcgaatta tttttgatgg cgttggaatt
//
