LOCUS       sequence-426257-        5602 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of GST-tagged mouse GTSF1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5602)
  AUTHORS   Arif A, Bailey S, Izumi N, Anzelon TA, Ozata DM, Andersson C, 
            Gainetdinov I, MacRae IJ, Tomari Y, Zamore PD
  TITLE     GTSF1 accelerates target RNA cleavage by PIWI-clade Argonaute 
            proteins.
  JOURNAL   Nature. 2022 Aug;608(7923):618-625. doi: 10.1038/s41586-022-05009-0.
            Epub 2022 Jun 30.
  PUBMED    35772669
REFERENCE   2  (bases 1 to 5602)
  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/s41586-022-05009-0"; journalName: "Nature"; date: 
            "2022-08"; volume: "608"; issue: "7923"; pages: "618-625"
COMMENT     Sequence Label: pColdI-GST-mGTSF1
FEATURES             Location/Qualifiers
     source          1..5602
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(719..1801)
                     /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"
     primer_bind     1763..1782
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1802..1879)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        1979..2045
                     /gene="cspA"
                     /label=cspA promoter
                     /note="promoter of the E. coli cold shock protein cspA gene
                     (Mitta et al., 1997)"
     protein_bind    2048..2064
                     /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)."
     5'UTR           2086..2219
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             2220..2234
                     /codon_start=1
                     /gene="cspA"
                     /product="translation enhancing element for E. coli (Qing 
                     et al., 2004)"
                     /label=TEE
                     /translation="MNHKV"
     CDS             2235..2252
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2253..2264
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2268..2921
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2879..2901
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2928..2951
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     3'UTR           3527..3671
                     /gene="cspA"
                     /label=cspA 3'UTR
                     /note="3'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     primer_bind     complement(3712..3734)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3834..3853
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(3870..4325)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4007..4028)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4219..4238
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4425..4529
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4530..4598,4599..5390)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4748..4767)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5561..5602,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
      661 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
      721 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
      781 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
      841 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
      901 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
      961 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     1021 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     1081 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     1141 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     1201 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     1261 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     1321 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     1381 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     1441 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     1501 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     1561 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     1621 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     1681 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     1741 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     1801 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     1861 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     1921 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgtggcc
     1981 gattaatcat aaatatgaaa aataattgtt gcatcacccg ccaatgcgtg gcttaatgca
     2041 catcaaattg tgagcggata acaatttgat gtgctagcgc atatccagtg tagtaaggca
     2101 agtcccttca agagttatcg ttgatacccc tcgtagtgca cattccttta acgcttcaaa
     2161 atctgtaaag cacgccatat cgccgaaagg cacacttaat tattaagagg taatacacca
     2221 tgaatcacaa agtgcatcat catcatcatc atatcgaagg taggcacatg tcccctatac
     2281 taggttattg gaaaattaag ggccttgtgc aacccactcg acttcttttg gaatatcttg
     2341 aagaaaaata tgaagagcat ttgtatgagc gcgatgaagg tgataaatgg cgaaacaaaa
     2401 agtttgaatt gggtttggag tttcccaatc ttccttatta tattgatggt gatgttaaat
     2461 taacacagtc tatggccatc atacgttata tagctgacaa gcacaacatg ttgggtggtt
     2521 gtccaaaaga gcgtgcagag atttcaatgc ttgaaggagc ggttttggat attagatacg
     2581 gtgtttcgag aattgcatat agtaaagact ttgaaactct caaagttgat tttcttagca
     2641 agctacctga aatgctgaaa atgttcgaag atcgtttatg tcataaaaca tatttaaatg
     2701 gtgatcatgt aacccatcct gacttcatgt tgtatgacgc tcttgatgtt gttttataca
     2761 tggacccaat gtgcctggat gcgttcccaa aattagtttg ttttaaaaaa cgtattgaag
     2821 ctatcccaca aattgataag tacttgaaat ccagcaagta tatagcatgg cctttgcagg
     2881 gctggcaagc cacgtttggt ggtggcgacc atcctccaaa atcggatctg gaagttctgt
     2941 tccagggccc gggacatatg gagctcggta ccctcgaggg atccatggaa gacacttaca
     3001 tcgactccct ggaccctgaa aagttattac agtgccccta tgataaaaat caccagatca
     3061 gggcctgcag gtttccttat catcttatca agtgcagaaa gaatcatcct gatgtcgcaa
     3121 acaaattggc tacttgtccc ttcaatgctc ggcatcaggt tccccgggct gagatcagtc
     3181 atcacatctc gagctgtgat gacaagagct gtattgagca ggatgtggtc aaccaaacca
     3241 ggaaccttgg acaagagact ctggctgaga gcacatggca gtgccctcct tgtgatgaag
     3301 actgggataa agatttgtgg gaacagacta gcaccccatt tgtctggggc acagccagtt
     3361 tctgtggcaa caacagtcct gcaaataaca tagttatgga acataagagt aaccttgctt
     3421 caggcatgcg agttcccaag tctctgccct atgttttgcc atggaaaaac aatggaaatg
     3481 cacagtaagg aattcaagct tgtcgacctg cagtctagat aggtaatctc tgcttaaaag
     3541 cacagaatct aagatccctg ccatttggcg gggatttttt tatttgtttt caggaaataa
     3601 ataatcgatc gcgtaataaa atctattatt atttttgtga agaataaatt tgggtgcaat
     3661 gagaatgcgc aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     3721 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     3781 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc
     3841 agagcagatt gtactgagag tgcaccataa aattgtaaac gttaatattt tgttaaaatt
     3901 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     3961 cccttataaa tcaaaagaat agcccgagat agggttgagt gttgttccag tttggaacaa
     4021 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     4081 cgatggccca ctacgtgaac catcacccaa atcaagtttt ttggggtcga ggtgccgtaa
     4141 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     4201 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     4261 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     4321 cgcgtactat ggttgctttg acgtatgcgg tgtgaaatac cgcacagatg cgtaaggaga
     4381 aaataccgca tcaggcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     4441 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     4501 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     4561 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     4621 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     4681 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     4741 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     4801 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     4861 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     4921 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     4981 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5041 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     5101 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     5161 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     5221 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     5281 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     5341 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     5401 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     5461 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     5521 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     5581 gcgtaatctg ctgcttgcaa ac
//
