LOCUS       sequence-256096-        5371 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes VSVG tagged GSK3(1-97) for purification by GST affinity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5371)
  AUTHORS   Smolko CM, Janes KA
  TITLE     An ultrasensitive fiveplex activity assay for cellular kinases.
  JOURNAL   Sci Rep. 2019 Dec 19;9(1):19409. doi: 10.1038/s41598-019-55998-8.
  PUBMED    31857650
REFERENCE   2  (bases 1 to 5371)
  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: "Sci Rep."; 
            date: "2019-12-19"; volume: "9(1):19409. doi"; pages: " 
            10.1038/s41598-019-55998-8"
COMMENT     Sequence Label: pGEX-4T-1-3xVSVG-GSK3(1-97)
FEATURES             Location/Qualifiers
     source          1..5371
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     47..64
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        137..214
                     /gene="lacI"
                     /label=lacI promoter
     CDS             215..1297
                     /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     complement(234..253)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1346..1351,1352..1369,1370..1376)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1384..1400
                     /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)."
     primer_bind     1408..1424
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1408..1424
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1420..1947
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(1440..1457)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1440..1456)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1449..1471)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(2049..2054,2055..2070,2071..2077)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2085..2101
                     /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)."
     CDS             2124..2777
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2735..2757
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2784..2801
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2805..2837
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     CDS             2838..2870
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     CDS             2871..2903
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     primer_bind     complement(3287..3309)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3454..3472)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3540..3644
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3645..3713,3714..4505)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3863..3882)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4676..5264
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5165..5184
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
       61 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
      121 acaccgcata aattccgaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata
      181 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
      241 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
      301 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
      361 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
      421 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
      481 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
      541 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
      601 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
      661 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
      721 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
      781 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca
      841 aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
      901 catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat
      961 ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc
     1021 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
     1081 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
     1141 gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
     1201 cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     1261 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt
     1321 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     1381 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacggattca
     1441 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     1501 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     1561 ccttcccaac agttgcgcag cctgaatggc gaatggcgct ttgcctggtt tccggcacca
     1621 gaagcggtgc cggaaagctg gctggagtgc gatcttcctg aggccgatac tgtcgtcgtc
     1681 ccctcaaact ggcagatgca cggttacgat gcgcccatct acaccaacgt gacctatccc
     1741 attacggtca atccgccgtt tgttcccacg gagaatccga cgggttgtta ctcgctcaca
     1801 tttaatgttg atgaaagctg gctacaggaa ggccagacgc gaattatttt tgatggcgtt
     1861 ggaattagct tatcgactgc acggtgcacc aatgcttctg gcgtcaggca gccatcggaa
     1921 gctgtggtat ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac
     1981 tcccgttctg gataatgttt tttgcgccga catcataacg gttctggcaa atattctgaa
     2041 atgagctgtt gacaattaat catcggctcg tataatgtgt ggaattgtga gcggataaca
     2101 atttcacaca ggaaacagta ttcatgtccc ctatactagg ttattggaaa attaagggcc
     2161 ttgtgcaacc cactcgactt cttttggaat atcttgaaga aaaatatgaa gagcatttgt
     2221 atgagcgcga tgaaggtgat aaatggcgaa acaaaaagtt tgaattgggt ttggagtttc
     2281 ccaatcttcc ttattatatt gatggtgatg ttaaattaac acagtctatg gccatcatac
     2341 gttatatagc tgacaagcac aacatgttgg gtggttgtcc aaaagagcgt gcagagattt
     2401 caatgcttga aggagcggtt ttggatatta gatacggtgt ttcgagaatt gcatatagta
     2461 aagactttga aactctcaaa gttgattttc ttagcaagct acctgaaatg ctgaaaatgt
     2521 tcgaagatcg tttatgtcat aaaacatatt taaatggtga tcatgtaacc catcctgact
     2581 tcatgttgta tgacgctctt gatgttgttt tatacatgga cccaatgtgc ctggatgcgt
     2641 tcccaaaatt agtttgtttt aaaaaacgta ttgaagctat cccacaaatt gataagtact
     2701 tgaaatccag caagtatata gcatggcctt tgcagggctg gcaagccacg tttggtggtg
     2761 gcgaccatcc tccaaaatcg gatctggttc cgcgtggatc tatgtacacc gatatagaga
     2821 tgaacaggct gggaaagtac accgatatag agatgaacag gctgggaaag tacaccgata
     2881 tagagatgaa caggctggga aagaagcttg gatccatgag cggcggcggg ccttcgggag
     2941 gcggccctgg gggctcgggc agggcgcgga ctagctcgtt cgcggagccc ggcggcggag
     3001 gcggaggagg cggcggcggc cccggaggct cggcctccgg cccaggcggc accggcggcg
     3061 gaaaggcatc tgtcggggcc atgggtgggg gcgtcggggc ctcgagctcc gggggtggac
     3121 ccggcggcag cggcggagga ggcagcggag gccccggcgc aggcactagc ttcccgccgc
     3181 ccggggtgaa gctgggccgt gacagcgaat tcccgggtcg actcgagcgg ccgcatcgtg
     3241 actgactgac gatctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     3301 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     3361 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     3421 atagcggagt gtataattct tgaagacgaa agggcctcgt gatacgccta tttttatagg
     3481 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     3541 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     3601 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     3661 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     3721 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     3781 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     3841 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     3901 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     3961 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     4021 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     4081 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     4141 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     4201 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     4261 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     4321 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     4381 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     4441 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     4501 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     4561 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     4621 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4681 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4741 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4801 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     4861 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4921 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4981 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     5041 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5101 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5161 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5221 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     5281 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     5341 cccctgattc tgtggataac cgtattaccg c
//
