LOCUS       sequence-238810-        6925 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express His-tagged PKM2 (R399E) 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-R399E
FEATURES             Location/Qualifiers
     source          1..6925
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             81..98
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             108..125
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1771..1788
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1841..1859)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1855..1902
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1939..2394
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2026..2045)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2236..2257
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2486..3301)
                     /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(2486..3301)
                     /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     3209..3228
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3293..3312
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3423..4011
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3912..3931
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4165..4182
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4197..4336
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4262..4281)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4422..4444
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4438..4629)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5438..6520)
                     /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     6482..6501
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6521..6598)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6804..6823
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(6907..6925,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6907..6925
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatataccat gggcagcagc catcatcatc atcatcacag cagcggcctg gtgccgcgcg
      121 gcagccatat gtcgaagccc catagtgaag ccgggactgc cttcattcag acccagcagc
      181 tgcacgcagc catggctgac acattcctgg agcacatgtg ccgcctggac attgattcac
      241 cacccatcac agcccggaac actggcatca tctgtaccat tggcccagct tcccgatcag
      301 tggagacgtt gaaggagatg attaagtctg gaatgaatgt ggctcgtctg aacttctctc
      361 atggaactca tgagtaccat gcggagacca tcaagaatgt gcgcacagcc acggaaagct
      421 ttgcttctga ccccatcctc taccggcccg ttgctgtggc tctagacact aaaggacctg
      481 agatccgaac tgggctcatc aagggcagcg gcactgcaga ggtggagctg aagaagggag
      541 ccactctcaa aatcacgctg gataacgcct acatggaaaa gtgtgacgag aacatcctgt
      601 ggctggacta caagaacatc tgcaaggtgg tggaagtggg cagcaagatc tacgtggatg
      661 atgggcttat ttctctccag gtgaagcaga aaggtgccga cttcctggtg acggaggtgg
      721 aaaatggtgg ctccttgggc agcaagaagg gtgtgaacct tcctggggct gctgtggact
      781 tgcctgctgt gtcggagaag gacatccagg atctgaagtt tggggtcgag caggatgttg
      841 atatggtgtt tgcgtcattc atccgcaagg catctgatgt ccatgaagtt aggaaggtcc
      901 tgggagagaa gggaaagaac atcaagatta tcagcaaaat cgagaatcat gagggggttc
      961 ggaggtttga tgaaatcctg gaggccagtg atgggatcat ggtggctcgt ggtgatctag
     1021 gcattgagat tcctgcagag aaggtcttcc ttgctcagaa gatgatgatt ggacggtgca
     1081 accgagctgg gaagcctgtc atctgtgcta ctcagatgct ggagagcatg atcaagaagc
     1141 cccgccccac tcgggctgaa ggcagtgatg tggccaatgc agtcctggat ggagccgact
     1201 gcatcatgct gtctggagaa acagccaaag gggactatcc tctggaggct gtgcgcatgc
     1261 agcacctgat tgcccgtgag gcagaggctg ccatctacca cttgcaatta tttgaggaac
     1321 tcgagcgcct ggcgcccatt accagcgacc ccacagaagc caccgccgtg ggtgccgtgg
     1381 aggcctcctt caagtgctgc agtggggcca taatcgtcct caccaagtct ggcaggtctg
     1441 ctcaccaggt ggccagatac cgcccacgtg cccccatcat tgctgtgacc cggaatcccc
     1501 agacagctcg tcaggcccac ctgtaccgtg gcatcttccc tgtgctgtgc aaggacccag
     1561 tccaggaggc ctgggctgag gacgtggacc tccgggtgaa ctttgccatg aatgttggca
     1621 aggcccgagg cttcttcaag aagggagatg tggtcattgt gctgaccgga tggcgccctg
     1681 gctccggctt caccaacacc atgcgtgttg ttcctgtgcc gtgaggatcc gaattcgagc
     1741 tccgtcgaca agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct
     1801 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     1861 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     1921 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     1981 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2041 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2101 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2161 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2221 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2281 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     2341 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     2401 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2461 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca
     2521 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     2581 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     2641 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     2701 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
     2761 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     2821 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa
     2881 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     2941 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     3001 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     3061 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     3121 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     3181 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     3241 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca
     3301 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc
     3361 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     3421 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     3481 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     3541 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     3601 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     3661 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     3721 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     3781 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     3841 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     3901 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     3961 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4021 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4081 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     4141 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     4201 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     4261 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     4321 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     4381 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4441 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     4501 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     4561 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     4621 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     4681 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     4741 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     4801 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     4861 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     4921 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     4981 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     5041 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc
     5101 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     5161 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     5221 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     5281 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     5341 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     5401 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5461 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5521 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     5581 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     5641 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     5701 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     5761 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     5821 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     5881 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     5941 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     6001 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     6061 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     6121 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     6181 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     6241 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     6301 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     6361 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     6421 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     6481 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     6541 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     6601 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     6661 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     6721 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     6781 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     6841 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg
     6901 cgaaattaat acgactcact atagg
//
