LOCUS       sequence-256108-        5665 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes FLAG tagged human HSP27 for purification by GST affinity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5665)
  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 5665)
  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-1-3xFLAG-HSP27
FEATURES             Location/Qualifiers
     source          1..5665
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     54..71
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        144..221
                     /gene="lacI"
                     /label=lacI promoter
     CDS             222..1304
                     /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(241..260)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1353..1358,1359..1376,1377..1383)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1391..1407
                     /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     1415..1431
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1415..1431
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1427..1954
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(1447..1464)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1447..1463)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1456..1478)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(2056..2061,2062..2077,2078..2084)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2092..2108
                     /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             2131..2784
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2742..2764
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2791..2808
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2812..2883
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     primer_bind     complement(3588..3610)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3755..3773)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3841..3945
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3946..4014,4015..4806)
                     /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(4164..4183)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4977..5565
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5466..5485
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
       61 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
      121 gtatttcaca ccgcataaat tccgacacca tcgaatggcg caaaaccttt cgcggtatgg
      181 catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat
      241 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg
      301 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt
      361 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg
      421 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
      481 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct
      541 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc
      601 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat
      661 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaagacggta
      721 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
      781 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc
      841 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc
      901 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg
      961 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg
     1021 atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa
     1081 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac
     1141 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa
     1201 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     1261 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     1321 gtaagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     1381 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     1441 ggattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     1501 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     1561 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgctttg cctggtttcc
     1621 ggcaccagaa gcggtgccgg aaagctggct ggagtgcgat cttcctgagg ccgatactgt
     1681 cgtcgtcccc tcaaactggc agatgcacgg ttacgatgcg cccatctaca ccaacgtgac
     1741 ctatcccatt acggtcaatc cgccgtttgt tcccacggag aatccgacgg gttgttactc
     1801 gctcacattt aatgttgatg aaagctggct acaggaaggc cagacgcgaa ttatttttga
     1861 tggcgttgga attagcttat cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc
     1921 atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa
     1981 ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt ctggcaaata
     2041 ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga attgtgagcg
     2101 gataacaatt tcacacagga aacagtattc atgtccccta tactaggtta ttggaaaatt
     2161 aagggccttg tgcaacccac tcgacttctt ttggaatatc ttgaagaaaa atatgaagag
     2221 catttgtatg agcgcgatga aggtgataaa tggcgaaaca aaaagtttga attgggtttg
     2281 gagtttccca atcttcctta ttatattgat ggtgatgtta aattaacaca gtctatggcc
     2341 atcatacgtt atatagctga caagcacaac atgttgggtg gttgtccaaa agagcgtgca
     2401 gagatttcaa tgcttgaagg agcggttttg gatattagat acggtgtttc gagaattgca
     2461 tatagtaaag actttgaaac tctcaaagtt gattttctta gcaagctacc tgaaatgctg
     2521 aaaatgttcg aagatcgttt atgtcataaa acatatttaa atggtgatca tgtaacccat
     2581 cctgacttca tgttgtatga cgctcttgat gttgttttat acatggaccc aatgtgcctg
     2641 gatgcgttcc caaaattagt ttgttttaaa aaacgtattg aagctatccc acaaattgat
     2701 aagtacttga aatccagcaa gtatatagca tggcctttgc agggctggca agccacgttt
     2761 ggtggtggcg accatcctcc aaaatcggat ctggttccgc gtggatctat ggattacaag
     2821 gatgacgacg ataaggatta caaggatgac gacgataagg attacaagga tgacgacgat
     2881 aagggatcca tgaccgagcg ccgcgtcccc ttctcgctcc tgcggggccc cagctgggac
     2941 cccttccgcg actggtaccc gcatagccgc ctcttcgacc aggccttcgg gctgccccgg
     3001 ctgccggagg agtggtcgca gtggttaggc ggcagcagct ggccaggcta cgtgcgcccc
     3061 ctgccccccg ccgccatcga gagccccgca gtggccgcgc ccgcctacag ccgcgcgctc
     3121 agccggcaac tcagcagcgg ggtctcggag atccggcaca ctgcggaccg ctggcgcgtg
     3181 tccctggatg tcaaccactt cgccccggac gagctgacgg tcaagaccaa ggatggcgtg
     3241 gtggagatca ccggcaagca cgaggagcgg caggacgagc atggctacat ctcccggtgc
     3301 ttcacgcgga aatacacgct gccccccggt gtggacccca cccaagtttc ctcctccctg
     3361 tcccctgagg gcacactgac cgtggaggcc cccatgccca agctagccac gcagtccaac
     3421 gagatcacca tcccagtcac cttcgagtcg cgggcccagc ttgggggccc agaagctgca
     3481 aaatccgatg agactgccgc caagtaagaa ttcccgggtc gactcgagcg gccgcatcgt
     3541 gactgactga cgatctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     3601 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     3661 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
     3721 gatagcggag tgtataattc ttgaagacga aagggcctcg tgatacgcct atttttatag
     3781 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     3841 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     3901 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     3961 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     4021 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     4081 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     4141 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
     4201 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     4261 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     4321 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     4381 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     4441 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca
     4501 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     4561 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     4621 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     4681 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     4741 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     4801 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     4861 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     4921 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4981 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5041 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5101 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     5161 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5221 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5281 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     5341 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5401 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5461 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5521 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5581 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     5641 tcccctgatt ctgtggataa ccgta
//
