LOCUS       sequence-426278-        5553 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of GST-tagged GTSF2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5553)
  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 5553)
  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-mGTSF2
FEATURES             Location/Qualifiers
     source          1..5553
                     /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           3478..3622
                     /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(3663..3685)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3785..3804
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(3821..4276)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3958..3979)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4170..4189
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4376..4480
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4481..4549,4550..5341)
                     /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(4699..4718)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5512..5553,1..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 atccgagttg gaatccatag
     3001 agctatgtcc ttatgatcca aaccacagga tacctgccag caggttacag taccatctgg
     3061 catcatgcaa aaagaaaaat ccaaagatag ctaaaaagat ggctaactgc aaatataatg
     3121 cttgtcatgt ggtcccaatc aaaaggctca aggaacatga ggccaactgt atcaacagga
     3181 ctgcagtgga tgatgagcca cttaaccttc aaaagattac tcacccagtt tttgaagaaa
     3241 atgaaaactt ctccagtgct gggagtcaat ttcctgaccc tgatgtctgg aacgtggatc
     3301 atgcgcatca ttttccttca tttgttcttg agacttttgc tccaaagaag ctagtttgtg
     3361 agagtgactc gagagaccta caagaggcta tggctgataa gcatcctaac agcttcaagt
     3421 cttggggaag agggcagaaa aactgaaagc ttgtcgacct gcagtctaga taggtaatct
     3481 ctgcttaaaa gcacagaatc taagatccct gccatttggc ggggattttt ttatttgttt
     3541 tcaggaaata aataatcgat cgcgtaataa aatctattat tatttttgtg aagaataaat
     3601 ttgggtgcaa tgagaatgcg caggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
     3661 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     3721 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
     3781 tatgcggcat cagagcagat tgtactgaga gtgcaccata aaattgtaaa cgttaatatt
     3841 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     3901 atcggcaaaa tcccttataa atcaaaagaa tagcccgaga tagggttgag tgttgttcca
     3961 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     4021 gtctatcagg gcgatggccc actacgtgaa ccatcaccca aatcaagttt tttggggtcg
     4081 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     4141 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     4201 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     4261 ccgctacagg gcgcgtacta tggttgcttt gacgtatgcg gtgtgaaata ccgcacagat
     4321 gcgtaaggag aaaataccgc atcaggcgtc aggtggcact tttcggggaa atgtgcgcgg
     4381 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     4441 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     4501 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     4561 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     4621 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     4681 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     4741 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     4801 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     4861 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     4921 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     4981 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     5041 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     5101 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     5161 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     5221 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     5281 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     5341 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     5401 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     5461 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5521 tttttttctg cgcgtaatct gctgcttgca aac
//
