LOCUS       sequence-239334-        6925 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express His-tagged PKM2 (H391Y) protein in bacteria..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6925)
  AUTHORS   Chen TJ, Wang HJ, Liu JS, Cheng HH, Hsu SC, Wu MC, Lu CH, Wu YF, Wu 
            JW, Liu YY, Kung HJ, Wang WC
  TITLE     Mutations in the PKM2 exon-10 region are associated with reduced 
            allostery and increased nuclear translocation.
  JOURNAL   Commun Biol. 2019 Mar 15;2:105. doi: 10.1038/s42003-019-0343-4. 
            eCollection 2019.
  PUBMED    30911680
REFERENCE   2  (bases 1 to 6925)
  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: "Commun 
            Biol."; date: "2019-03-15"; volume: "2:105. doi"; pages: " 
            10.1038/s42003-019-0343-4. eCollection 2019"
COMMENT     Sequence Label: pET28a-PKM2-H391Y
FEATURES             Location/Qualifiers
     source          1..6925
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             15..37
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             56..73
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             83..100
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1746..1763
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1816..1834)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1830..1877
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1914..2369
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2001..2020)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2211..2232
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2461..3276)
                     /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(2461..3276)
                     /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     3184..3203
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3268..3287
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3398..3986
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3887..3906
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4140..4157
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4172..4311
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4237..4256)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4397..4419
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4413..4604)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5413..6495)
                     /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     6457..6476
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6496..6573)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6779..6798
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6882..6901
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6882..6900
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6901..6925
                     /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)."
ORIGIN
        1 cctctagaaa taattttgtt taactttaag aaggagatat accatgggca gcagccatca
       61 tcatcatcat cacagcagcg gcctggtgcc gcgcggcagc catatgtcga agccccatag
      121 tgaagccggg actgccttca ttcagaccca gcagctgcac gcagccatgg ctgacacatt
      181 cctggagcac atgtgccgcc tggacattga ttcaccaccc atcacagccc ggaacactgg
      241 catcatctgt accattggcc cagcttcccg atcagtggag acgttgaagg agatgattaa
      301 gtctggaatg aatgtggctc gtctgaactt ctctcatgga actcatgagt accatgcgga
      361 gaccatcaag aatgtgcgca cagccacgga aagctttgct tctgacccca tcctctaccg
      421 gcccgttgct gtggctctag acactaaagg acctgagatc cgaactgggc tcatcaaggg
      481 cagcggcact gcagaggtgg agctgaagaa gggagccact ctcaaaatca cgctggataa
      541 cgcctacatg gaaaagtgtg acgagaacat cctgtggctg gactacaaga acatctgcaa
      601 ggtggtggaa gtgggcagca agatctacgt ggatgatggg cttatttctc tccaggtgaa
      661 gcagaaaggt gccgacttcc tggtgacgga ggtggaaaat ggtggctcct tgggcagcaa
      721 gaagggtgtg aaccttcctg gggctgctgt ggacttgcct gctgtgtcgg agaaggacat
      781 ccaggatctg aagtttgggg tcgagcagga tgttgatatg gtgtttgcgt cattcatccg
      841 caaggcatct gatgtccatg aagttaggaa ggtcctggga gagaagggaa agaacatcaa
      901 gattatcagc aaaatcgaga atcatgaggg ggttcggagg tttgatgaaa tcctggaggc
      961 cagtgatggg atcatggtgg ctcgtggtga tctaggcatt gagattcctg cagagaaggt
     1021 cttccttgct cagaagatga tgattggacg gtgcaaccga gctgggaagc ctgtcatctg
     1081 tgctactcag atgctggaga gcatgatcaa gaagccccgc cccactcggg ctgaaggcag
     1141 tgatgtggcc aatgcagtcc tggatggagc cgactgcatc atgctgtctg gagaaacagc
     1201 caaaggggac tatcctctgg aggctgtgcg catgcagcac ctgattgccc gtgaggcaga
     1261 ggctgccatc tactatttgc aattatttga ggaactccgc cgcctggcgc ccattaccag
     1321 cgaccccaca gaagccaccg ccgtgggtgc cgtggaggcc tccttcaagt gctgcagtgg
     1381 ggccataatc gtcctcacca agtctggcag gtctgctcac caggtggcca gataccgccc
     1441 acgtgccccc atcattgctg tgacccggaa tccccagaca gctcgtcagg cccacctgta
     1501 ccgtggcatc ttccctgtgc tgtgcaagga cccagtccag gaggcctggg ctgaggacgt
     1561 ggacctccgg gtgaactttg ccatgaatgt tggcaaggcc cgaggcttct tcaagaaggg
     1621 agatgtggtc attgtgctga ccggatggcg ccctggctcc ggcttcacca acaccatgcg
     1681 tgttgttcct gtgccgtgag gatccgaatt cgagctccgt cgacaagctt gcggccgcac
     1741 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
     1801 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     1861 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
     1921 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     1981 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2041 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2101 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2161 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2221 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2281 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2341 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     2401 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
     2461 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     2521 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     2581 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     2641 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     2701 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     2761 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     2821 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     2881 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     2941 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     3001 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     3061 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     3121 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     3181 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     3241 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     3301 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact
     3361 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3421 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3481 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3541 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3601 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3661 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3721 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3781 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     3841 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     3901 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     3961 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4021 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4081 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4141 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     4201 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     4261 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     4321 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4381 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     4441 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     4501 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     4561 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     4621 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     4681 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     4741 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     4801 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     4861 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     4921 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     4981 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     5041 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     5101 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     5161 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     5221 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     5281 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     5341 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     5401 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5461 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     5521 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     5581 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     5641 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     5701 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     5761 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     5821 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     5881 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     5941 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     6001 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     6061 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     6121 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     6181 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     6241 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     6301 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     6361 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     6421 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     6481 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     6541 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     6601 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     6661 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     6721 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     6781 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     6841 tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg
     6901 ggaattgtga gcggataaca attcc
//
