LOCUS       sequence-212053-        5163 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 5163)
  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 5163)
  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..5163
                     /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"
     CDS             815..832
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     promoter        complement(905..923)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(906..923)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(981..999)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      995..1042
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1278..1299)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    1418..1439
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(1452..2534)
                     /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     2496..2515
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2535..2612)
                     /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     2758..2777
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2933..2952)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3052..3074
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3112..3130)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3198..3302
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3303..3371,3372..4163)
                     /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      4334..4922
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4823..4842
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5076..5093
                     /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 ccgaggggat ccatggatat
      841 cgaattcgtc gacctcgagg gatccgggcc ctctagatgc ggccgcatgc ataagcttga
      901 gtattctata gtgtcaccta aatcccagct tgatccggct gctaacaaag cccgaaagga
      961 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     1021 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggataac ctggcgtaat
     1081 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     1141 aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tatttgttaa atcagctcat
     1201 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     1261 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     1321 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacagctt gcatgcctgc
     1381 aggtcggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     1441 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     1501 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     1561 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     1621 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttgac
     1681 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca
     1741 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     1801 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     1861 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     1921 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     1981 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     2041 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     2101 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     2161 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     2221 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     2281 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     2341 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     2401 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     2461 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     2521 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     2581 ccgcgaaagg ttttgcacca ttcgatggtg tcgggtaccg agctcgaatt tacgtgaacc
     2641 atcacccaaa tcaagttttt tgcggtcgag gtgccgtaaa gctctaaatc ggaaccctaa
     2701 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     2761 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     2821 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcgcctg atgcggtatt
     2881 ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct
     2941 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     3001 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     3061 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     3121 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     3181 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     3241 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     3301 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     3361 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     3421 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     3481 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtcat acactattat
     3541 cccgtattga cgccgggcaa gagcaactcg gtcgccgggc gcggtattct cagaatgact
     3601 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     3661 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     3721 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     3781 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     3841 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     3901 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     3961 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     4021 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     4081 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     4141 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     4201 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     4261 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4321 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4381 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4441 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     4501 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4561 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4621 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4681 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4741 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4801 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4861 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4921 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     4981 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     5041 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     5101 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     5161 gat
//
