LOCUS       sequence-289325-        5999 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Bacterial Expression of HsTNRC6Biso1_1379-1723.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5999)
  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 5999)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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_1379-1723_L
FEATURES             Location/Qualifiers
     source          1..5999
                     /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"
     primer_bind     complement(2049..2071)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2216..2234)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2302..2406
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2407..2475,2476..3267)
                     /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(2625..2644)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3438..4026
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3927..3946
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4180..4197
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4270..4347
                     /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             4348..5430
                     /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(4367..4386)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5443..5464
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5479..5484,5485..5502,5503..5509)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5517..5533
                     /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     5541..5557
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5541..5557
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5553..5999,1..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(5573..5590)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5573..5589)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5582..5604)
                     /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 atccaaaaca
      961 ttgaccctga atctgacccc tatgtcaccc caggaagtgt gctggggggt acagccacat
     1021 ctcccattgt agatactgac caccaactgc tgcgggataa caccacaggg tctaattctt
     1081 ccctcaacac ctcgctgcct tcacctggtg cctggcccta cagtgcctct gacaactcct
     1141 ttaccaacgt tcatagcact tcagcaaagt tccctgatta caaatcaaca tggtccccag
     1201 atcccatagg acacaacccc actcatctct ccaacaagat gtggaaaaac catatttcct
     1261 ccaggaacac tgcaccgctg ccccgcccac ctcctggtct gaccaacccc aaaccatcat
     1321 ctccctggag cagcacagca ccccgatcag tcagggggtg ggggacacag gactcacggc
     1381 tcgcctcggc ctctacctgg agtgatggtg gctcagttcg tcctagttac tggctggttc
     1441 ttcacaatct caccccacag attgatgggt caaccttgag aacgatctgc atgcagcatg
     1501 gcccactgct gacattccat ctgaatctaa cccagggcac tgccctgatc cgatacagca
     1561 ccaaacagga ggcggccaag gcccaaactg cactgcacat gtgtgtgttg ggaaacacta
     1621 ccatccttgc tgagtttgcc actgatgatg aagtcagccg ctttctggca caagctcagc
     1681 cccctacacc tgcagcaacc ccaagtgcgc cagctgcggg gtggcagtcg ctggagaccg
     1741 gccagaacca gtcagatccc gtgggacctg ctctgaatct ttttggtggg tccactgggc
     1801 tcgggcagtg gagcagcagc gctggtggca gcagcggggc cgatcttgct ggcgcttcat
     1861 tgtgggggcc cccaaactat tcttctagct tatggggagt cccaacggtg gaagatcccc
     1921 ataggatggg cagccctgct cctttactac ctggtgacct tctgggagga gggtcggatt
     1981 caatctgagc ggccgcatcg tgactgactg acgatctgcc tcgcgcgttt cggtgatgac
     2041 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     2101 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca
     2161 gccatgaccc agtcacgtag cgatagcgga gtgtataatt cttgaagacg aaagggcctc
     2221 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     2281 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     2341 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     2401 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     2461 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     2521 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     2581 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     2641 ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     2701 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     2761 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     2821 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     2881 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     2941 acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     3001 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     3061 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     3121 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     3181 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     3241 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     3301 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     3361 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3421 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3481 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3541 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     3601 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3661 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3721 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3781 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     3841 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3901 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3961 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4021 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4081 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4141 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4201 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4261 ataaattccg acaccatcga atggtgcaaa acctttcgcg gtatggcatg atagcgcccg
     4321 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag
     4381 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
     4441 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
     4501 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
     4561 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
     4621 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
     4681 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
     4741 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
     4801 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
     4861 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
     4921 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
     4981 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
     5041 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
     5101 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg
     5161 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgtcaaccac catcaaacag
     5221 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
     5281 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
     5341 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     5401 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     5461 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     5521 gagcggataa caatttcaca caggaaacag ctatgaccat gattacggat tcactggccg
     5581 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     5641 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     5701 aacagttgcg cagcctgaat ggcgaatggc gctttgcctg gtttccggca ccagaagcgg
     5761 tgccggaaag ctggctggag tgcgatcttc ctgaggccga tactgtcgtc gtcccctcaa
     5821 actggcagat gcacggttac gatgcgccca tctacaccaa cgtaacctat cccattacgg
     5881 tcaatccgcc gtttgttccc acggagaatc cgacgggttg ttactcgctc acatttaatg
     5941 ttgatgaaag ctggctacag gaaggccaga cgcgaattat ttttgatggc gttggaatt
//
