LOCUS       sequence-220183-        5185 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as Thrombin and TEV 
            cleavable N-terminal 6His-tag-GST fusions .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5185)
  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 5185)
  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: pGAT3
FEATURES             Location/Qualifiers
     source          1..5185
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     80..99
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        80..98
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    98..122
                     /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             135..157
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             177..194
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             213..863
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPP"
     primer_bind     824..846
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             894..911
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             918..938
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        complement(1014..1032)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1015..1032)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1090..1108)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1104..1151
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1387..1408)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1558..2640)
                     /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     2602..2621
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2641..2718)
                     /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     2861..2880
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3034..3053)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3153..3175
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3213..3231)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3299..3403
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3404..3472,3473..4264)
                     /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(3622..3641)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4435..5023
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4924..4943
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(5177..5185,1..9)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
       61 attcattaat gcagctgatt aatacgactc actataggga attgtgagcg gataacaatt
      121 cctctagaaa taattttgtt taactttaag aaggagatat atccatgaac accattcatc
      181 accatcacca tcacaacact agtagcaatt ccatgtcccc tatactaggt tattggaaaa
      241 ttaagggcct tgtgcaaccc actcgacttc ttttggaata tcttgaagaa aaatatgaag
      301 agcatttgta tgagcgcgat gaaggtgata aatggcgaaa caaaaagttt gaattgggtt
      361 tggagtttcc caatcttcct tattatattg atggtgatgt taaattaaca cagtctatgg
      421 ccatcatacg ttatatagct gacaagcaca acatgttggg tggttgtcca aaagagcgtg
      481 cagagatttc aatgcttgaa ggagcggttt tggatattag atacggtgtt tcgagaattg
      541 catatagtaa agactttgaa actctcaaag ttgattttct tagcaagcta cctgaaatgc
      601 tgaaaatgtt cgaagatcgt ttatgtcata aaacatattt aaatggtgat catgtaaccc
      661 atcctgactt catgttgtat gacgctcttg atgttgtttt atacatggac ccaatgtgcc
      721 tggatgcgtt cccaaaatta gtttgtttta aaaaacgtat tgaagctatc ccacaaattg
      781 ataagtactt gaaatccagc aagtatatag catggccttt gcagggctgg caagccacgt
      841 ttggtggtgg cgaccatcct ccaactagtg gatctggtgg tggtggcggc cgcctggttc
      901 cgaggggatc catgtctgaa aacctgtact tccagggttc catggatatc gaattcgtcg
      961 acctcgaggg atccgggccc tctagatgcg gccgcatgca taagcttgag tattctatag
     1021 tgtcacctaa atcccagctt gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     1081 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     1141 ggggtttttt gctgaaagga ggaactatat ccggataacc tggcgtaata gcgaagaggc
     1201 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga aattgtaagc
     1261 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     1321 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     1381 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     1441 cgaaaaaccg tctatcaggg cgatggccca cagcttgcat gcctgcaggt cggaagcata
     1501 aagtgtaaag cctggggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     1561 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     1621 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     1681 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     1741 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     1801 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     1861 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     1921 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     1981 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     2041 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     2101 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     2161 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     2221 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     2281 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     2341 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     2401 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     2461 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     2521 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     2581 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     2641 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     2701 gcaccattcg atggtgtcgg gtaccgagct cgaattgtga accatcaccc taatcaagtt
     2761 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     2821 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     2881 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     2941 cgcttaatgc gccgctacag ggcgcgtcct gatgcggtat tttctcctta cgcatctgtg
     3001 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3061 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     3121 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     3181 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     3241 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     3301 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     3361 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     3421 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     3481 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     3541 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     3601 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     3661 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     3721 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     3781 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     3841 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     3901 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     3961 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     4021 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     4081 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     4141 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     4201 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     4261 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     4321 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     4381 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     4441 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     4501 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     4561 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     4621 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     4681 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4741 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4801 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4861 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4921 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4981 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     5041 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     5101 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     5161 ccgaacgacc gagcgcagcg agtca
//
