LOCUS       sequence-212054-        5193 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 5193)
  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 5193)
  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..5193
                     /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"
     CDS             839..859
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        complement(935..953)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(936..953)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1011..1029)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1025..1072
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1308..1329)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    1448..1469
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(1482..2564)
                     /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     2526..2545
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2565..2642)
                     /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     2788..2807
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2963..2982)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3082..3104
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3142..3160)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3228..3332
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3333..3401,3402..4193)
                     /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      4364..4952
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4853..4872
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5106..5123
                     /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 ccatgtctga
      841 aaacctgtac ttccagggtt ccatggatat cgaattcgtc gacctcgagg gatccgggcc
      901 ctctagatgc ggccgcatgc ataagcttga gtattctata gtgtcaccta aatcccagct
      961 tgatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     1021 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     1081 aggaactata tccggataac ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     1141 caacagttgc gcagcctgaa tggcgaatgg aaattgtaaa cgttaatatt ttgttaaaat
     1201 tcgcgttaaa tatttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     1261 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     1321 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     1381 gcgatggccc actacagctt gcatgcctgc aggtcggaag cataaagtgt aaagcctggg
     1441 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     1501 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     1561 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     1621 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     1681 aggcgaaaat cctgtttgat ggtggttgac ggcgggatat aacatgagct gtcttcggta
     1741 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     1801 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     1861 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     1921 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     1981 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     2041 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     2101 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     2161 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     2221 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     2281 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     2341 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     2401 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     2461 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     2521 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     2581 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcacca ttcgatggtg
     2641 tcgggtaccg agctcgaatt tacgtgaacc atcacccaaa tcaagttttt tgcggtcgag
     2701 gtgccgtaaa gctctaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     2761 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     2821 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     2881 gctacagggc gcgtcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     2941 accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     3001 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3061 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3121 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     3181 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     3241 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     3301 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     3361 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     3421 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     3481 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     3541 ttaaagttct gctatgtcat acactattat cccgtattga cgccgggcaa gagcaactcg
     3601 gtcgccgggc gcggtattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     3661 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     3721 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     3781 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     3841 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     3901 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     3961 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     4021 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     4081 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     4141 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     4201 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     4261 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     4321 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     4381 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     4441 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4501 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4561 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4621 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4681 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4741 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4801 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4861 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4921 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4981 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     5041 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     5101 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     5161 ccgcgcgttg gccgattcat taatgcagct gat
//
