LOCUS       sequence-220166-        5134 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 5134)
  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 5134)
  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..5134
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     58..77
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        58..76
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    76..100
                     /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             113..135
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             155..172
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             191..841
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPP"
     primer_bind     802..824
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     promoter        complement(941..959)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(942..959)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1017..1035)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1031..1078
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1314..1335)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1485..2567)
                     /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     2529..2548
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2568..2645)
                     /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(2961..2980)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3080..3102
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3140..3158)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3226..3330
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3331..3399,3400..4191)
                     /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(3549..3568)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4362..4950
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4851..4870
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5104..5121
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctgattaa
       61 tacgactcac tatagggaat tgtgagcgga taacaattcc tctagaaata attttgttta
      121 actttaagaa ggagatatat ccatgaacac cattcatcac catcaccatc acaacactag
      181 tagcaattcc atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac
      241 tcgacttctt ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga
      301 aggtgataaa tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta
      361 ttatattgat ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga
      421 caagcacaac atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg
      481 agcggttttg gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac
      541 tctcaaagtt gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt
      601 atgtcataaa acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga
      661 cgctcttgat gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt
      721 ttgttttaaa aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa
      781 gtatatagca tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc
      841 aactagtgga tctggtggtg gtggcggccg cctggttccg aggcctatcg gatccatggc
      901 tcgagttccc gggcaagctt gagtgactga gcttgagtat tctatagtgt cacctaaatc
      961 ccagcttgat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     1021 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1081 gaaaggagga actatatccg gataacctgg cgtaatagcg aagaggcccg caccgatcgc
     1141 ccttcccaac agttgcgcag cctgaatggc gaatggaaat tgtaagcgtt aatattttgt
     1201 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     1261 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     1321 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     1381 atcagggcga tggcccacag cttgcatgcc tgcaggtcgg aagcataaag tgtaaagcct
     1441 ggggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     1501 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     1561 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     1621 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     1681 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     1741 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     1801 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     1861 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     1921 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     1981 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     2041 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     2101 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     2161 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     2221 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     2281 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     2341 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     2401 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     2461 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     2521 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     2581 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgca ccattcgatg
     2641 gtgtcgggta ccgagctcga attgtgaacc atcaccctaa tcaagttttt tggggtcgag
     2701 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     2761 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     2821 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     2881 gctacagggc gcgtcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     2941 cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     3001 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3061 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3121 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     3181 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3241 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3301 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     3361 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     3421 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     3481 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     3541 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     3601 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     3661 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     3721 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     3781 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     3841 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     3901 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     3961 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     4021 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     4081 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     4141 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     4201 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     4261 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4321 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4381 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4441 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4501 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4561 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4621 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4681 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     4741 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     4801 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4861 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4921 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4981 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     5041 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     5101 cgcagcgagt cagtgagcga ggaagcggaa gagc
//
