LOCUS       sequence-289472-        5765 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of HsTNRC6Biso1_1361-1621.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5765)
  AUTHORS   Huntzinger E, Braun JE, Heimstadt S, Zekri L, Izaurralde E
  TITLE     Two PABPC1-binding sites in GW182 proteins promote miRNA-mediated 
            gene silencing.
  JOURNAL   EMBO J. 2010 Dec 15;29(24):4146-60. doi: 10.1038/emboj.2010.274
  PUBMED    21063388
REFERENCE   2  (bases 1 to 5765)
  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: 
            "10.1038/emboj.2010"; journalName: "EMBO J"; date: "2010-12-15- 15";
            volume: "29"; issue: "24"; pages: "4146-60"
COMMENT     Sequence Label: pGEX-6P-1-HsTNRC6Biso1_1361-1621-6His_N
FEATURES             Location/Qualifiers
     source          1..5765
                     /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"
     CDS             1734..1751
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1815..1837)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1982..2000)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2068..2172
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2173..2241,2242..3033)
                     /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(2391..2410)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3204..3792
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3693..3712
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3946..3963
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4036..4113
                     /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             4114..5196
                     /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(4133..4152)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5209..5230
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5245..5250,5251..5268,5269..5275)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5283..5299
                     /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     5307..5323
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5307..5323
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5319..5765,1..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(5339..5356)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5339..5355)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5348..5370)
                     /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 tccagcaatg
      961 ccagttggcc tccagaattc caaccaggag tgccatggaa aggtatccaa aacattgacc
     1021 ctgaatctga cccctatgtc accccaggaa gtgtgctggg gggtacagcc acatctccca
     1081 ttgtagatac tgaccaccaa ctgctgcggg ataacaccac agggtctaat tcttccctca
     1141 acacctcgct gccttcacct ggtgcctggc cctacagtgc ctctgacaac tcctttacca
     1201 acgttcatag cacttcagca aagttccctg attacaaatc aacatggtcc ccagatccca
     1261 taggacacaa ccccactcat ctctccaaca agatgtggaa aaaccatatt tcctccagga
     1321 acactacacc gctgccccgc ccacctcctg gtctgaccaa ccccaaacca tcatctccct
     1381 ggagcagcac agcaccccga tcagtcaggg ggtgggggac acaggactca cggctcgcct
     1441 cggcctctac ctggagtgat ggtggctcag ttcgtcctag ttactggctg gttcttcaca
     1501 atctcacccc acagattgat gggtcaacct tgagaacgat ctgcatgcag catggcccac
     1561 tgctgacatt ccatctgaat ctaacccagg gcactgccct gatccgatac agcaccaaac
     1621 aggaggcggc caaggcccaa actgcactgc acatgtgtgt gttgggaaac actaccatcc
     1681 ttgctgagtt tgccactgat gatgaagtca gccgctttct ggcacaagct cagcatcacc
     1741 atcaccatca ctaagcggcc gcatcgtgac tgactgacga tctgcctcgc gcgtttcggt
     1801 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     1861 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     1921 ggcgcagcca tgacccagtc acgtagcgat agcggagtgt ataattcttg aagacgaaag
     1981 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     2041 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     2101 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     2161 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     2221 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     2281 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     2341 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     2401 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     2461 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     2521 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     2581 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     2641 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     2701 gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     2761 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     2821 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     2881 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     2941 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     3001 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     3061 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     3121 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     3181 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3241 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3301 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     3361 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     3421 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     3481 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     3541 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3601 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3661 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3721 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3781 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3841 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     3901 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     3961 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4021 caccgcataa attccgacac catcgaatgg tgcaaaacct ttcgcggtat ggcatgatag
     4081 cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc
     4141 gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac
     4201 gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc
     4261 aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc
     4321 agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa
     4381 ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg
     4441 gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat
     4501 gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat
     4561 gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg
     4621 ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta
     4681 agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa
     4741 attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc
     4801 atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg
     4861 gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg
     4921 gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtc aaccaccatc
     4981 aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag
     5041 ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc
     5101 ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     5161 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     5221 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     5281 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acggattcac
     5341 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     5401 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     5461 cttcccaaca gttgcgcagc ctgaatggcg aatggcgctt tgcctggttt ccggcaccag
     5521 aagcggtgcc ggaaagctgg ctggagtgcg atcttcctga ggccgatact gtcgtcgtcc
     5581 cctcaaactg gcagatgcac ggttacgatg cgcccatcta caccaacgta acctatccca
     5641 ttacggtcaa tccgccgttt gttcccacgg agaatccgac gggttgttac tcgctcacat
     5701 ttaatgttga tgaaagctgg ctacaggaag gccagacgcg aattattttt gatggcgttg
     5761 gaatt
//
