LOCUS       sequence-255082-        5645 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes VSVG tagged GSK3(1-97) for purification by nickel affinity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5645)
  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 5645)
  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: pET-29b-3xVSVG-GSK3(1-97)
FEATURES             Location/Qualifiers
     source          1..5645
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     38..57
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        38..56
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    57..81
                     /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             96..118
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             129..161
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     CDS             162..194
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     CDS             195..227
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     CDS             516..536
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             543..560
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(613..631)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      627..674
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      711..1166
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(798..817)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1008..1029
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1258..2073)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1258..2073)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1981..2000
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2065..2084
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2195..2783
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2684..2703
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2937..2954
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2969..3108
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3034..3053)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3194..3216
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3210..3401)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4210..5292)
                     /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     5254..5273
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5293..5370)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5576..5595
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gcgtagagga tcgagatcga tctcgatccc gcgaaattaa tacgactcac tataggggaa
       61 ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata
      121 tacatatgta caccgatata gagatgaaca ggctgggaaa gtacaccgat atagagatga
      181 acaggctggg aaagtacacc gatatagaga tgaacaggct gggaaagagc ggcggcgggc
      241 cttcgggagg cggccctggg ggctcgggca gggcgcggac tagctcgttc gcggagcccg
      301 gcggcggagg cggaggaggc ggcggcggcc ccggaggctc ggcctccggc ccaggcggca
      361 ccggcggcgg aaaggcatct gtcggggcca tgggtggggg cgtcggggcc tcgagctccg
      421 ggggtggacc cggcggcagc ggcggaggag gcagcggagg ccccggcgca ggcactagct
      481 tcccgccgcc cggggtgaag ctgggccgtg acagcgagaa cctatacttc caaggggtcg
      541 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
      601 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
      661 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
      721 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
      781 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
      841 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
      901 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
      961 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     1021 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     1081 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     1141 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     1201 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     1261 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     1321 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     1381 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     1441 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     1501 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     1561 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     1621 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     1681 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     1741 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     1801 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     1861 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     1921 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     1981 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     2041 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     2101 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     2161 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     2221 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     2281 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     2341 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     2401 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     2461 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     2521 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     2581 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     2641 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     2701 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     2761 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     2821 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     2881 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     2941 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     3001 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3061 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     3121 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     3181 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     3241 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     3301 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     3361 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     3421 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     3481 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     3541 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     3601 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     3661 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     3721 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     3781 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     3841 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc
     3901 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     3961 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     4021 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     4081 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     4141 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     4201 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4261 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     4321 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     4381 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     4441 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc
     4501 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     4561 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     4621 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     4681 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     4741 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     4801 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     4861 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     4921 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     4981 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     5041 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     5101 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     5161 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     5221 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     5281 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     5341 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     5401 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     5461 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     5521 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     5581 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     5641 gtccg
//
