LOCUS       sequence-370087-        7012 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the human liver pyruvate kinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7012)
  AUTHORS   McFarlane JS, Ronnebaum TA, Meneely KM, Chilton A, Fenton AW, Lamb 
            AL
  TITLE     Changes in the allosteric site of human liver pyruvate kinase upon 
            activator binding include the breakage of an intersubunit cation-pi 
            bond.
  JOURNAL   Acta Crystallogr F Struct Biol Commun. 2019 Jun 1;75(Pt 6):461-469. 
            doi: 10.1107/S2053230X19007209. Epub 2019 Jun 10.
  PUBMED    31204694
REFERENCE   2  (bases 1 to 7012)
  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: "Acta 
            Crystallogr F Struct Biol Commun."; date: "2019-06-1"; volume: 
            "75(Pt 6):461-469. doi"; pages: " 10.1107/S2053230X19007209. Epub 
            2019 Jun 10"
COMMENT     Sequence Label: pET15TV-L-hlPYK WT
FEATURES             Location/Qualifiers
     source          1..7012
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    63..202
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(217..234)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(388..976)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(468..487)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1147..1938,1939..2007))
                     /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     1770..1789
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2008..2112)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2180..2198
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(2225..2230,2231..2247,2248..2253)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(2428..2475)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2471..2489
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     gap             2862..3075
                     /estimated_length=214
     CDS             complement(4180..4200)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(4213..4230)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             4249..4271
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4286..4310
                     /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     complement(4310..4329)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4311..4329)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     gap             4333..4391
                     /estimated_length=59
     primer_bind     complement(4413..4432)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4638..4715
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4716..5798
                     /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="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAXX
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQXXXXVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4735..4754)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
       61 ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg
      121 catcrgagca gattgtactg agagtgcacc attgcggtgt gaaataccgc acagatgcgt
      181 aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
      241 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
      301 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
      361 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
      421 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
      481 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
      541 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
      601 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
      661 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
      721 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
      781 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
      841 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
      901 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
      961 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     1021 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     1081 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     1141 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     1201 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     1261 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     1321 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     1381 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     1441 gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     1501 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     1561 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     1621 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     1681 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     1741 gagttgctct tgcccggcgt caacacggga taataccgcg ccacatagca gaactttaaa
     1801 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     1861 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     1921 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     1981 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     2041 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     2101 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat
     2161 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc aagaattctc
     2221 atgtttgaca gcttatcatc gataagcttt aatgcggtag tttatcacag ttaaattgct
     2281 aacgcagtca ggcaccgtgt atgaaatcta acaatgcgct catcgtcatc cycggcaccg
     2341 tsaccctgga tgctgtaggc ataggcttgg ttatgccggt actgccgggc ctcttgcggg
     2401 atatccggat atagttcctc ctttcagcaa aaaacccctc aagacccgtt tagaggcccc
     2461 aaggggttat gctagttatt gctcagcggt ggcagcagcc aactcagctt cctttcgggc
     2521 tttgttagca gccggatcct cgagtcagct aatgctcaga acacgcatga tgttggtata
     2581 gccgctaccc ggacgccaac cggtaaccac gatcaccaga tcgccaacac gcagaaaacc
     2641 acgcagtttg ccgctttcaa taccgaactg cacacgacgg tcaacatcgt ccgcccagat
     2701 cgcctccggc ggttcacgat acagcagcgg aaacacacca cggcacaggt gaacttgacg
     2761 cgccgcctgc gcgctacggg tcaccgcaat aaccgccgca cgcggacggt aacggctcag
     2821 cagttgcgcg ctacgaccgg tggtggtcag aacaatgayy gnnnnnnnnn nnnnnnnnnn
     2881 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
     2941 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
     3001 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
     3061 nnnnnnnnnn nnnnnrccgt ccagcaccgc gttcgcarca tcgctggtct ccgcacgggt
     3121 cggacgcggc ttggtaatca tgctttccag catttgggtc gcgcaaacca ccggcttgcc
     3181 cgccaggttg caacggccga tcatcatttt ctgcgccaga aaaaccttct ccgccggaat
     3241 ttcgatgccc aggtcaccac gcgccaccat aataccatcg ctcacttcca ggatttcgtc
     3301 gaaacgtttc acgccctcgt ggttttcaat tttgctaatg atcttgatgc cgtgaccctc
     3361 cggacccagc gccgcacgca ccgccgcaac atcgctcgcc ttacgcacaa agctcgcgaa
     3421 cacgatgtca acgccgtgct caacaccaaa acgcagatca cgcacgtctt gttcgctcag
     3481 acccggcagg tcaacctgcg cacccggcag gttcacgcct ttacggctac ccagaacgcc
     3541 accgttctcc acttgggtaa ccaggccttc cgggccgatc ttctgaacca ccaggctgat
     3601 cagaccatcg tcaatataga tacggccacc caccggaacc acacgaacga tgttcgggta
     3661 atccacccaa acggtgttcg cgttaccacg ggtacggaac gccgggtcca cggtaaccag
     3721 cacttggctg cctttcacca gctcaacttc gctctccggg ccaccctgca gaataccggt
     3781 acggatttcc gggcccttgg tatccagcgc aatcgcaacc ggacgatagc tcagcgggct
     3841 acccgcaaag ctctccaccg cttcacgaac gttcgcgatg ctctccgcgt ggtattcgtg
     3901 gctaccgtgg ctgaagttca gacgcgcaat gttcatgccc kstttgatca tctccttcag
     3961 acgttcaacg ctacggctcg ccggaccgat ggtcgcaatg atgctggtgc tacgcgccgc
     4021 caccggctcg ctatcaatgt ccagcaggca caggtgttcc aggaaggtgt ccgccatcgc
     4081 cgccggcagc tgttgttgct gaaagaacgc ggtacccagc tcttgggtca gctgcgccac
     4141 gctcgcacga cgcaggtaac ccgccggacc ttccatatgg ccctggaaat acaagttttc
     4201 tctgccgctg ctgtgatgat gatgatgatg gctgctgccc atggtatatc tccttcttaa
     4261 agttaaacaa aattatttct agaggggaat tgttatccgc tcacaattcc cctatagtga
     4321 gtcgtattaa ttnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
     4381 nnnnnnnnnn nccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga
     4441 agatcgggct cgccacttcg ggctcatgag cgctkgtttc ggcgtgggta tggtggcagg
     4501 ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc
     4561 ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg
     4621 agagcgtcga gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat
     4681 agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg
     4741 tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc
     4801 acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggasmkg aattacattc
     4861 ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct
     4921 ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc
     4981 aacygnnnnn nnnsgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag
     5041 cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg
     5101 atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg
     5161 atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac
     5221 tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat
     5281 taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc
     5341 aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa
     5401 ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga
     5461 tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct
     5521 cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca
     5581 tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc
     5641 agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca
     5701 ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     5761 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt
     5821 tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc
     5881 agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt
     5941 atcatgcaac tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc
     6001 tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc
     6061 ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt
     6121 atcgccggca tggcggccga cgcgctgggc tacgtcttgc tggcgttcgc gacgcgaggc
     6181 tggatggcct tccccattat gattcttctc gcttccggcg gcatcgggat gcccgcgttg
     6241 caggccatgc tgtccaggca ggtagatgac gaccatcagg gacmgcttca aggatcgctc
     6301 gcggctctta ccagcctaac ttcgatcatt ggaccgctga tcgtcacggc gatttatgcc
     6361 gcctcggcga gcacatggaa cgggttggca tggattgtag gcgccgccct ataccttgtc
     6421 tgcctccccg cgttgcgtcg cggtgcatgg agccgggcca cctcgaccyg aatggaagcc
     6481 ggcggcacct cgctaacgga ttcaccactc caagaattgg agccaatcaa ttcttgcgga
     6541 gaactgtgaa tgcgcaaacc aacccttggc agaacatatc catcgcgtcc gccatctcca
     6601 gcagccgcac gcggcgcatc tcgggcagcg ttgggtcctg gccacgggtg cgcatgatcg
     6661 tgctcctgtc gttgaggacc cggctaggct ggcggggttg ccttactggt tagcagaatg
     6721 aatcaccgat acgcgagcga acgtgaagcg actgctgctg caaaacgtct gcgacctgag
     6781 caacaacatg aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag
     6841 cgccctgcac cattatgttc cggatctgca tcgcaggatg ctgctggcta ccctgtggaa
     6901 cacctacatc tgtattaacg aagcgctggc attgaccctg agtgattttt ctctggtccc
     6961 gccgcatcca taccgccagt tgtttaccct cacaacgttc cagtaaccgg gc
//
