LOCUS       sequence-238806-        6955 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express His-tagged PKM2 (G415R) protein in bacteria..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6955)
  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 6955)
  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-G415R
FEATURES             Location/Qualifiers
     source          1..6955
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1..23
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             42..59
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             69..86
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1762..1779
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1832..1850)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1846..1893
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1930..2385
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2017..2036)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2227..2248
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2477..3292)
                     /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(2477..3292)
                     /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     3200..3219
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3284..3303
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3414..4002
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3903..3922
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4156..4173
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4188..4327
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4253..4272)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4413..4435
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4429..4620)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5429..6511)
                     /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     6473..6492
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6512..6589)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6795..6814
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6898..6917
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6898..6916
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6917..6941
                     /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 tttgtttaac tttaagaagg agatatacca tgggcagcag ccatcatcat catcatcaca
       61 gcagcggcct ggtgccgcgc ggcagccata tgtttggctt cactatgggg cttcgacata
      121 tgtcgaagcc ccatagtgaa gccgggactg ccttcattca gacccagcag ctgcacgcag
      181 ccatggctga cacattcctg gagcacatgt gccgcctgga cattgattca ccacccatca
      241 cagcccggaa cactggcatc atctgtacca ttggcccagc ttcccgatca gtggagacgt
      301 tgaaggagat gattaagtct ggaatgaatg tggctcgtct gaacttctct catggaactc
      361 atgagtacca tgcggagacc atcaagaatg tgcgcacagc cacggaaagc tttgcttctg
      421 accccatcct ctaccggccc gttgctgtgg ctctagacac taaaggacct gagatccgaa
      481 ctgggctcat caagggcagc ggcactgcag aggtggagct gaagaaggga gccactctca
      541 aaatcacgct ggataacgcc tacatggaaa agtgtgacga gaacatcctg tggctggact
      601 acaagaacat ctgcaaggtg gtggaagtgg gcagcaagat ctacgtggat gatgggctta
      661 tttctctcca ggtgaagcag aaaggtgccg acttcctggt gacggaggtg gaaaatggtg
      721 gctccttggg cagcaagaag ggtgtgaacc ttcctggggc tgctgtggac ttgcctgctg
      781 tgtcggagaa ggacatccag gatctgaagt ttggggtcga gcaggatgtt gatatggtgt
      841 ttgcgtcatt catccgcaag gcatctgatg tccatgaagt taggaaggtc ctgggagaga
      901 agggaaagaa catcaagatt atcagcaaaa tcgagaatca tgagggggtt cggaggtttg
      961 atgaaatcct ggaggccagt gatgggatca tggtggctcg tggtgatcta ggcattgaga
     1021 ttcctgcaga gaaggtcttc cttgctcaga agatgatgat tggacggtgc aaccgagctg
     1081 ggaagcctgt catctgtgct actcagatgc tggagagcat gatcaagaag ccccgcccca
     1141 ctcgggctga aggcagtgat gtggccaatg cagtcctgga tggagccgac tgcatcatgc
     1201 tgtctggaga aacagccaaa ggggactatc ctctggaggc tgtgcgcatg cagcacctga
     1261 ttgcccgtga ggcagaggct gccatctacc acttgcaatt atttgaggaa ctccgccgcc
     1321 tggcgcccat taccagcgac cccacagaag ccaccgccgt gcgtgccgtg gaggcctcct
     1381 tcaagtgctg cagtggggcc ataatcgtcc tcaccaagtc tggcaggtct gctcaccagg
     1441 tggccagata ccgcccacgt gcccccatca ttgctgtgac ccggaatccc cagacagctc
     1501 gtcaggccca cctgtaccgt ggcatcttcc ctgtgctgtg caaggaccca gtccaggagg
     1561 cctgggctga ggacgtggac ctccgggtga actttgccat gaatgttggc aaggcccgag
     1621 gcttcttcaa gaagggagat gtggtcattg tgctgaccgg atggcgccct ggctccggct
     1681 tcaccaacac catgcgtgtt gttcctgtgc cgtgaggatc cgaattcgag ctccgtcgac
     1741 aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
     1801 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     1861 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     1921 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     1981 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2041 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2101 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2161 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2221 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     2281 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     2341 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     2401 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2461 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     2521 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     2581 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     2641 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     2701 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     2761 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     2821 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     2881 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     2941 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     3001 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     3061 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     3121 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     3181 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     3241 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     3301 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     3361 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3421 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3481 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3541 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     3601 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     3661 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3721 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3781 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3841 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3901 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3961 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4021 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4081 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4141 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     4201 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     4261 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     4321 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4381 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4441 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     4501 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     4561 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     4621 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     4681 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     4741 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     4801 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     4861 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     4921 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     4981 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     5041 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     5101 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     5161 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     5221 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     5281 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     5341 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     5401 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5461 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5521 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     5581 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     5641 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     5701 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     5761 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     5821 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     5881 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     5941 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     6001 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     6061 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     6121 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     6181 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     6241 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     6301 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     6361 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     6421 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     6481 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     6541 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     6601 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     6661 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     6721 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     6781 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     6841 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
     6901 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataat
//
