LOCUS       sequence-403121-        7340 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 7340)
  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 7340)
  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..7340
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(662..1744)
                     /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     1706..1725
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1745..1822)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2028..2047
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2131..2150
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2131..2149
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2150..2174
                     /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             2189..2211
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2230..2247
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2260..2280
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3971..3989)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3985..4032
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4207..4212,4213..4229,4230..4235))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4262..4280)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4348..4452
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4453..4521,4522..5313)
                     /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     complement(4671..4690)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5484..6072
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5973..5992
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6226..6243
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6258..6397
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6323..6342)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6483..6505
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6499..6690)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ggcccggctc catgcaccgc gacgcaacgc ggggaggcag acaaggtata gggcggcgcc
       61 tacaatccat gccaacccgt tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat
      121 cagcggtcca atgatcgaag ttaggctggt aagagccgcg agcgatcctt gaagctgtcc
      181 ctgatggtcg tcatctacct gcctggacag catggcctgc aacgcgggca tcccgatgcc
      241 gccggaagcg agaagaatca taatggggaa ggccatccag cctcgcgtcg cgaacgccag
      301 caagacgtag cccagcgcgt cggccgccat gccggcgata atggcctgct tctcgccgaa
      361 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
      421 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
      481 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
      541 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
      601 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
      661 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
      721 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
      781 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
      841 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
      901 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
      961 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     1021 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     1081 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     1141 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     1201 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     1261 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     1321 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     1381 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     1441 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     1501 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     1561 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     1621 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     1681 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     1741 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     1801 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     1861 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     1921 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     1981 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     2041 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     2101 tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag
     2161 cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg
     2221 ggcagcagcc atcatcatca tcatcacagc agcggcagag aaaacttgta tttccagggc
     2281 catatggaag gtccggcggg ttacctgcgt cgtgcgagcg tggcgcagct gacccaagag
     2341 ctgggtaccg cgttctttca gcaacaacag ctgccggcgg cgatggcgga caccttcctg
     2401 gaacacctgt gcctgctgga cattgatagc gagccggtgg cggcgcgtag caccagcatc
     2461 attgcgacca tcggtccggc gagccgtagc gttgaacgtc tgaaggagat gatcaaagcg
     2521 ggcatgaaca ttgcgcgtct gaacttcagc cacggtagcc acgaatacca cgcggagagc
     2581 atcgcgaacg ttcgtgaagc ggtggagagc tttgcgggta gcccgctgag ctatcgtccg
     2641 gttgcgattg cgctggatac caagggcccg gaaatccgta ccggtattct gcagggtggc
     2701 ccggagagcg aagttgagct ggtgaaaggc agccaagtgc tggttaccgt ggacccggcg
     2761 ttccgtaccc gtggtaacgc gaacaccgtt tgggtggatt acccgaacat cgttcgtgtg
     2821 gttccggtgg gtggccgtat ctatattgac gatggtctga tcagcctggt ggttcagaag
     2881 atcggcccgg aaggcctggt tacccaagtg gagaacggtg gcgttctggg tagccgtaaa
     2941 ggcgtgaacc tgccgggtgc gcaggttgac ctgccgggtc tgagcgaaca agacgtgcgt
     3001 gatctgcgtt ttggtgttga gcacggcgtt gacatcgtgt tcgcgagctt tgtgcgtaag
     3061 gcgagcgatg ttgcggcggt gcgtgcggcg ctgggtccgg agggtcacgg catcaagatc
     3121 attagcaaaa ttgaaaacca cgagggcgtg aaacgtttcg acgaaatcct ggaagtgagc
     3181 gatggtatta tggtggcgcg tggtgacctg ggcatcgaaa ttccggcgga gaaggttttt
     3241 ctggcgcaga aaatgatgat cggccgttgc aacctggcgg gcaagccggt ggtttgcgcg
     3301 acccaaatgc tggaaagcat gattaccaag ccgcgtccga cccgtgcgga gaccagcgat
     3361 gttgcgaacg cggtgctgga cggtgcggat tgcatcatgc tgagcggcga aaccgcgaag
     3421 ggtaacttcc cggttgaggc ggtgaaaatg caacatgcga ttgcgcgtga agcggaggcg
     3481 gcggtgtacc accgtcaact gtttgaagaa ctgcgtcgtg cggcgccgct gagccgtgat
     3541 ccgaccgaag ttaccgcgat tggtgcggtt gaggcggcgt tcaagtgctg cgcggcggcg
     3601 atcattgttc tgaccaccac cggtcgtagc gcgcaactgc tgagccgtta ccgtccgcgt
     3661 gcggcggtta ttgcggtgac ccgtagcgcg caggcggcgc gtcaagttca cctgtgccgt
     3721 ggtgtgtttc cgctgctgta tcgtgaaccg ccggaggcga tctgggcgga cgatgttgac
     3781 cgtcgtgtgc agttcggtat tgaaagcggc aaactgcgtg gttttctgcg tgttggcgat
     3841 ctggtgatcg tggttaccgg ttggcgtccg ggtagcggct ataccaacat catgcgtgtt
     3901 ctgagcatta gctgactcga ggatccggct gctaacaaag cccgaaagga agctgagttg
     3961 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     4021 aggggttttt tgctgaaagg aggaactata tccggatatc ccgcaagagg cccggcagta
     4081 ccggcataac caagcctatg cctacagcat ccagggtgac ggtgccgagg atgacgatga
     4141 gcgcattgtt agatttcata cacggtgcct gactgcgtta gcaatttaac tgtgataaac
     4201 taccgcatta aagcttatcg atgataagct gtcaaacatg agaattcttg aagacgaaag
     4261 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     4321 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4381 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4441 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     4501 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     4561 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     4621 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     4681 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     4741 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     4801 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     4861 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     4921 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     4981 gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     5041 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     5101 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     5161 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     5221 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     5281 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     5341 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     5401 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5461 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5521 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     5581 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     5641 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     5701 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     5761 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     5821 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     5881 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     5941 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6001 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6061 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6121 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     6181 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     6241 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     6301 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     6361 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     6421 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     6481 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     6541 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     6601 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     6661 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     6721 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     6781 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     6841 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     6901 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     6961 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     7021 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     7081 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     7141 gatcatgcgc acccgtggcc aggacccaac gctgcccgag atgcgccgcg tgcggctgct
     7201 ggagatggcg gacgcgatgg atatgttctg ccaagggttg gtttgcgcat tcacagttct
     7261 ccgcaagaat tgattggctc caattcttgg agtggtgaat ccgttagcga ggtgccgccg
     7321 gcttccattc aggtcgaggt
//
