LOCUS       sequence-212052-        5142 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as Thrombin cleavable 
            6His-tag-GST fusions with a blunt end restriction site immediately 
            after thrombin recognition sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5142)
  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 5142)
  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: pGAT
FEATURES             Location/Qualifiers
     source          1..5142
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    19..43
                     /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             56..78
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             98..115
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             134..784
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPP"
     primer_bind     745..767
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     promoter        complement(884..902)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(885..902)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(960..978)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      974..1021
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1257..1278)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    1397..1418
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(1431..2513)
                     /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     2475..2494
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2514..2591)
                     /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     2737..2756
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2912..2931)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3031..3053
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3091..3109)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3177..3281
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3282..3350,3351..4142)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCHTLLSRIDAGQEQLGRRARYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      4313..4901
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4802..4821
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5055..5072
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa caccattcat caccatcacc atcacaacac
      121 tagtagcaat tccatgtccc ctatactagg ttattggaaa attaagggcc ttgtgcaacc
      181 cactcgactt cttttggaat atcttgaaga aaaatatgaa gagcatttgt atgagcgcga
      241 tgaaggtgat aaatggcgaa acaaaaagtt tgaattgggt ttggagtttc ccaatcttcc
      301 ttattatatt gatggtgatg ttaaattaac acagtctatg gccatcatac gttatatagc
      361 tgacaagcac aacatgttgg gtggttgtcc aaaagagcgt gcagagattt caatgcttga
      421 aggagcggtt ttggatatta gatacggtgt ttcgagaatt gcatatagta aagactttga
      481 aactctcaaa gttgattttc ttagcaagct acctgaaatg ctgaaaatgt tcgaagatcg
      541 tttatgtcat aaaacatatt taaatggtga tcatgtaacc catcctgact tcatgttgta
      601 tgacgctctt gatgttgttt tatacatgga cccaatgtgc ctggatgcgt tcccaaaatt
      661 agtttgtttt aaaaaacgta ttgaagctat cccacaaatt gataagtact tgaaatccag
      721 caagtatata gcatggcctt tgcagggctg gcaagccacg tttggtggtg gcgaccatcc
      781 tccaactagt ggatctggtg gtggtggcgg ccgcctggtt ccgaggccta tcggatccat
      841 ggctcgagtt cccgggcaag cttgagtgac tgagcttgag tattctatag tgtcacctaa
      901 atcccagctt gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
      961 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     1021 gctgaaagga ggaactatat ccggataacc tggcgtaata gcgaagaggc ccgcaccgat
     1081 cgcccttccc aacagttgcg cagcctgaat ggcgaatgga aattgtaaac gttaatattt
     1141 tgttaaaatt cgcgttaaat atttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     1201 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     1261 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     1321 tctatcaggg cgatggccca ctacagcttg catgcctgca ggtcggaagc ataaagtgta
     1381 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     1441 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     1501 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     1561 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     1621 tgccccagca ggcgaaaatc ctgtttgatg gtggttgacg gcgggatata acatgagctg
     1681 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
     1741 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     1801 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     1861 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     1921 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     1981 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     2041 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     2101 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     2161 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     2221 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     2281 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     2341 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     2401 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     2461 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     2521 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcaccat
     2581 tcgatggtgt cgggtaccga gctcgaattt acgtgaacca tcacccaaat caagtttttt
     2641 gcggtcgagg tgccgtaaag ctctaaatcg gaaccctaaa gggagccccc gatttagagc
     2701 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     2761 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     2821 taatgcgccg ctacagggcg cgtcgcctga tgcggtattt tctccttacg catctgtgcg
     2881 gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     2941 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     3001 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     3061 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     3121 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     3181 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     3241 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     3301 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     3361 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     3421 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     3481 tgagcacttt taaagttctg ctatgtcata cactattatc ccgtattgac gccgggcaag
     3541 agcaactcgg tcgccgggcg cggtattctc agaatgactt ggttgagtac tcaccagtca
     3601 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     3661 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     3721 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     3781 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     3841 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     3901 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     3961 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     4021 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     4081 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     4141 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     4201 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     4261 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     4321 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4381 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     4441 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     4501 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     4561 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     4621 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     4681 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     4741 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     4801 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     4861 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     4921 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     4981 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     5041 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     5101 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg at
//
