LOCUS       sequence-220181-        5155 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as Thrombin cleavable 
            N-terminal 6His-tag-GST fusions .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5155)
  AUTHORS   Peranen J, Rikkonen M, Hyvonen M, Kaariainen L
  TITLE     T7 vectors with modified T7lac promoter for expression of proteins 
            in Escherichia coli.
  JOURNAL   Anal Biochem. 1996 May 1;236(2):371-3.
  PUBMED    8660525
REFERENCE   2  (bases 1 to 5155)
  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"; journalName: "Anal 
            Biochem."; date: "1996-05-1"; volume: "236(2)"; pages: "371-3"
COMMENT     Sequence Label: pGAT2
FEATURES             Location/Qualifiers
     source          1..5155
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     72..89
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     160..179
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        160..178
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    178..202
                     /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)."
     RBS             215..237
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             257..274
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             293..943
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPP"
     primer_bind     904..926
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             974..991
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     promoter        complement(1064..1082)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1065..1082)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1140..1158)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1154..1201
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1437..1458)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1608..2690)
                     /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     2652..2671
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2691..2768)
                     /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."
     primer_bind     2911..2930
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3084..3103)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3203..3225
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3263..3281)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3349..3453
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3454..3522,3523..4314)
                     /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(3672..3691)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4485..5073
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4974..4993
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
       61 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
      121 ctctccccgc gcgttggccg attcattaat gcagctgatt aatacgactc actataggga
      181 attgtgagcg gataacaatt cctctagaaa taattttgtt taactttaag aaggagatat
      241 atccatgaac accattcatc accatcacca tcacaacact agtagcaatt ccatgtcccc
      301 tatactaggt tattggaaaa ttaagggcct tgtgcaaccc actcgacttc ttttggaata
      361 tcttgaagaa aaatatgaag agcatttgta tgagcgcgat gaaggtgata aatggcgaaa
      421 caaaaagttt gaattgggtt tggagtttcc caatcttcct tattatattg atggtgatgt
      481 taaattaaca cagtctatgg ccatcatacg ttatatagct gacaagcaca acatgttggg
      541 tggttgtcca aaagagcgtg cagagatttc aatgcttgaa ggagcggttt tggatattag
      601 atacggtgtt tcgagaattg catatagtaa agactttgaa actctcaaag ttgattttct
      661 tagcaagcta cctgaaatgc tgaaaatgtt cgaagatcgt ttatgtcata aaacatattt
      721 aaatggtgat catgtaaccc atcctgactt catgttgtat gacgctcttg atgttgtttt
      781 atacatggac ccaatgtgcc tggatgcgtt cccaaaatta gtttgtttta aaaaacgtat
      841 tgaagctatc ccacaaattg ataagtactt gaaatccagc aagtatatag catggccttt
      901 gcagggctgg caagccacgt ttggtggtgg cgaccatcct ccaactagtg gatctggtgg
      961 tggtggcggc cgcctggttc cgaggggatc catggatatc gaattcgtcg acctcgaggg
     1021 atccgggccc tctagatgcg gccgcatgca taagcttgag tattctatag tgtcacctaa
     1081 atcccagctt gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     1141 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     1201 gctgaaagga ggaactatat ccggataacc tggcgtaata gcgaagaggc ccgcaccgat
     1261 cgcccttccc aacagttgcg cagcctgaat ggcgaatgga aattgtaagc gttaatattt
     1321 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     1381 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     1441 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     1501 tctatcaggg cgatggccca cagcttgcat gcctgcaggt cggaagcata aagtgtaaag
     1561 cctggggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     1621 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     1681 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     1741 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     1801 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     1861 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     1921 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     1981 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     2041 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     2101 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     2161 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     2221 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     2281 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     2341 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     2401 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     2461 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     2521 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     2581 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     2641 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     2701 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcaccattcg
     2761 atggtgtcgg gtaccgagct cgaattgtga accatcaccc taatcaagtt ttttggggtc
     2821 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     2881 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     2941 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     3001 gccgctacag ggcgcgtcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     3061 caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     3121 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3181 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     3241 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     3301 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     3361 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     3421 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     3481 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     3541 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     3601 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     3661 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     3721 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     3781 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     3841 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     3901 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     3961 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     4021 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     4081 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     4141 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     4201 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     4261 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     4321 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     4381 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     4441 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4501 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4561 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4621 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4681 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4741 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4801 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4861 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4921 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4981 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     5041 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     5101 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctg
//
