LOCUS       sequence-441246-        6849 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible expression of rabbit muscle pyruvate kinase isoform 1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6849)
  AUTHORS   Swint-Kruse L, Dougherty LL, Page B, Wu T, O'Neil PT, Prasannan CB, 
            Timmons C, Tang Q, Parente DJ, Sreenivasan S, Holyoak T, Fenton AW
  TITLE     PYK-SubstitutionOME: an integrated database containing allosteric 
            coupling, ligand affinity and mutational, structural, pathological, 
            bioinformatic and computational information about pyruvate kinase 
            isozymes.
  JOURNAL   Database (Oxford). 2023 May 3;2023:baad030. doi: 
            10.1093/database/baad030.
  PUBMED    37171062
REFERENCE   2  (bases 1 to 6849)
  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: "Database 
            (Oxford)."; date: "2023-05-3"; volume: "2023:baad030. doi"; pages: "
            10.1093/database/baad030"
COMMENT     Sequence Label: pCDF-rM1PYK
FEATURES             Location/Qualifiers
     source          1..6849
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1347..1365
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1366..1390
                     /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)."
     CDS             1499..1543
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1581..1599)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1595..1642
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1813..2604)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(2605..2696)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(3757..4839)
                     /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     4801..4820
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4840..4917)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        4963..4981
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4982..5006
                     /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)."
     CDS             5056..5073
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5086..6186
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     6160..6183
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             6244..6534
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG"
ORIGIN
        1 cggcccgtcg cggtggctct ggacactaag ggacccgaga tccgaacggg gctcatcaag
       61 ggcagcggca cggcggaggt ggagctgaag aagggagcca cgctcaagat cacgctggac
      121 aacgcctaca tggagaagtg cgacgagaac atcctgtggc tggactacaa gaacatttgc
      181 aaggtggtgg acgtgggcag caaggtctac gtggacgatg gcctcatctc cctgcaggtg
      241 aagcagaaag gtcctgactt cctggtgacg gaggtggaga acggtggctt cttgggcagc
      301 aagaagggcg tgaacctccc cggggctgcg gtggacctgc ctgccgtgtc cgagaaggac
      361 atccaggatc tgaagttcgg cgtggagcag gacgtggaca tggtgtttgc ctctttcatc
      421 cgcaaggcgg ctgacgtcca tgaggtcagg aagatcctgg gagagaaagg gaagaacatc
      481 aagatcatca gcaaaattga gaaccacgag ggggtgcgca ggtttgatga gatcctggag
      541 gccagcgacg ggatcatggt ggctcgtggt gatcttggca ttgagattcc tgcagagaag
      601 gtcttccttg cccagaagat gataattggg cgatgcaacc gtgctgggaa gcctgtcatc
      661 tgtgccacgc agatgctgga gagcatgatc aagaagcccc gccccacccg cgccgagggc
      721 agtgacgtgg ccaacgcggt cctggatgga gccgactgca tcatgctgtc tggagagacg
      781 gccaagggcg actacccgct ggaggccgtc cgcatgcagc acctgatagc gcgcgaggct
      841 gaggcagcca tgttccaccg caagctgttc gaagaacttg cgcgcagctc cagtcactcc
      901 acagacctca tggaggccat ggccatgggc agcgtggagg cttcctataa gtgtttagcg
      961 gcagctttga tagttctgac ggagtctggc aggtctgctc accaagtggc ccggtaccgc
     1021 ccgcgcgccc ccatcattgc cgtgacacgg aaccaccaga ccgcccgcca ggcccacctg
     1081 taccgtggga tcttcccggt ggtgtgtaag gatccggtcc aggaggcctg ggctgaggat
     1141 gtcgacctcc gggtgaactt ggccatgaat gtcggcaagg cccgcggttt cttcaagaag
     1201 ggagatgtgg tcattgtgct caccggatgg cgccctggct ctggcttcac caacaccatg
     1261 cgcgtggtgc ccgtcccgtg agcggccgca taatgcttaa gtcgaacaga aagtaatcgt
     1321 attgtacacg gccgcataat cgaaattaat acgactcact ataggggaat tgtgagcgga
     1381 taacaattcc ccatcttagt atattagtta agtataagaa ggagatatac atatggcaga
     1441 tctcaattgg atatcggccg gccacgcgat cgctgacgtc ggtaccctcg agtctggtaa
     1501 agaaaccgct gctgcgaaat ttgaacgcca gcacatggac tcgtctacta gcgcagctta
     1561 attaacctag gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     1621 acgggtcttg aggggttttt tgctgaaacc tcaggcattt gagaagcaca cggtcacact
     1681 gcttccggta gtcaataaac cggtaaacca gcaatagaca taagcggcta tttaacgacc
     1741 ctgccctgaa ccgacgaccg ggtcatcgtg gccggatctt gcggcccctc ggcttgaacg
     1801 aattgttaga cattatttgc cgactacctt ggtgatctcg cctttcacgt agtggacaaa
     1861 ttcttccaac tgatctgcgc gcgaggccaa gcgatcttct tcttgtccaa gataagcctg
     1921 tctagcttca agtatgacgg gctgatactg ggccggcagg cgctccattg cccagtcggc
     1981 agcgacatcc ttcggcgcga ttttgccggt tactgcgctg taccaaatgc gggacaacgt
     2041 aagcactaca tttcgctcat cgccagccca gtcgggcggc gagttccata gcgttaaggt
     2101 ttcatttagc gcctcaaata gatcctgttc aggaaccgga tcaaagagtt cctccgccgc
     2161 tggacctacc aaggcaacgc tatgttctct tgcttttgtc agcaagatag ccagatcaat
     2221 gtcgatcgtg gctggctcga agatacctgc aagaatgtca ttgcgctgcc attctccaaa
     2281 ttgcagttcg cgcttagctg gataacgcca cggaatgatg tcgtcgtgca caacaatggt
     2341 gacttctaca gcgcggagaa tctcgctctc tccaggggaa gccgaagttt ccaaaaggtc
     2401 gttgatcaaa gctcgccgcg ttgtttcatc aagccttacg gtcaccgtaa ccagcaaatc
     2461 aatatcactg tgtggcttca ggccgccatc cactgcggag ccgtacaaat gtacggccag
     2521 caacgtcggt tcgagatggc gctcgatgac gccaactacc tctgatagtt gagtcgatac
     2581 ttcggcgatc accgcttccc tcatactctt cctttttcaa tattattgaa gcatttatca
     2641 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagc
     2701 tagctcactc ggtcgctacg ctccgggcgt gagactgcgg cgggcgctgc ggacacatac
     2761 aaagttaccc acagattccg tggataagca ggggactaac atgtgaggca aaacagcagg
     2821 gccgcgccgg tggcgttttt ccataggctc cgccctcctg ccagagttca cataaacaga
     2881 cgcttttccg gtgcatctgt gggagccgtg aggctcaacc atgaatctga cagtacgggc
     2941 gaaacccgac aggacttaaa gatccccacc gtttccggcg ggtcgctccc tcttgcgctc
     3001 tcctgttccg accctgccgt ttaccggata cctgttccgc ctttctccct tacgggaagt
     3061 gtggcgcttt ctcatagctc acacactggt atctcggctc ggtgtaggtc gttcgctcca
     3121 agctgggctg taagcaagaa ctccccgttc agcccgactg ctgcgcctta tccggtaact
     3181 gttcacttga gtccaacccg gaaaagcacg gtaaaacgcc actggcagca gccattggta
     3241 actgggagtt cgcagaggat ttgtttagct aaacacgcgg ttgctcttga agtgtgcgcc
     3301 aaagtccggc tacactggaa ggacagattt ggttgctgtg ctctgcgaaa gccagttacc
     3361 acggttaagc agttccccaa ctgacttaac cttcgatcaa accacctccc caggtggttt
     3421 tttcgaaggt taagtcagtt ggggaactgc ttaaccttcg atcaaaccac ctccccaggt
     3481 ggttttttcg tttacagggc aaaagattac gcgcagaaaa aaaggatctc aagaagatcc
     3541 tttgatcttt tctactgaac cgctctagat ttcagtgcaa tttatctctt caaatgtagc
     3601 acctgaagtc agccccatac gatataagtt gtaattctca tgttagtcat gccccgcgcc
     3661 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     3721 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3781 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3841 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     3901 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     3961 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     4021 gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     4081 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     4141 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     4201 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     4261 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     4321 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     4381 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     4441 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     4501 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     4561 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     4621 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     4681 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     4741 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     4801 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     4861 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     4921 gatctcgacg ctctccctta tgcgactcct gcattaggaa attaatacga ctcactatag
     4981 gggaattgtg agcggataac aattcccctg tagaaataat tttgtttaac tttaataagg
     5041 agatatacca tgggtcatca ccatcatcat cacgggtccg cgggcatgaa aatcgaagaa
     5101 ggtaaactgg taatctggat taacggcgat aaaggctata acggtctcgc tgaagtcggt
     5161 aagaaattcg agaaagatac cggaattaaa gtcaccgttg agcatccgga taaactggaa
     5221 gagaaattcc cacaggttgc ggcaactggc gatggccctg acattatctt ctgggcacac
     5281 gaccgctttg gtggctacgc tcaatctggc ctgttggctg aaatcacccc ggacaaagcg
     5341 ttccaggaca agctgtatcc gtttacctgg gatgccgtac gttacaacgg caagctgatt
     5401 gcttacccga tcgctgttga agcgttatcg ctgatttata acaaagatct gctgccgaac
     5461 ccgccaaaaa cctgggaaga gatcccggcg ctggataaag aactgaaagc gaaaggtaag
     5521 agcgcgctga tgttcaacct gcaagaaccg tacttcacct ggccgctgat tgctgctgac
     5581 gggggttatg cgttcaagta tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat
     5641 aacgctggcg cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa caaacacatg
     5701 aatgcagaca ccgattactc catcgcagaa gctgccttta ataaaggcga aacagcgatg
     5761 accatcaacg gcccgtgggc atggtccaac atcgacacca gcaaagtgaa ttatggtgta
     5821 acggtactgc cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca
     5881 ggtattaacg ccgccagtcc gaacaaagag ctggcaaaag agttcctcga aaactatctg
     5941 ctgactgatg aaggtctgga agcggttaat aaagacaaac cgctgggtgc cgtagcgctg
     6001 aagtcttacg aggaagagtt ggtgaaagat ccgcgtattg ccgccactat ggaaaacgcc
     6061 cagaaaggtg aaatcatgcc gaacatcccg cagatgtccg ctttctggta tgccgtgcgt
     6121 actgcggtga tcaacgccgc cagcggtcgt cagactgtcg atgaagccct gaaagacgcg
     6181 cagactaatt cgagctcgaa caacaacaac aataacaata acaacaacct cggggctagc
     6241 ggctcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct
     6301 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa
     6361 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg
     6421 gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcaggc ccctgaagat
     6481 ttggacatgg aggataacga tattattgag gctcatcgcg aacagattgg aggtatgtca
     6541 aagtcccaca gtgaagccgg gtctgccttc atccagacgc agcagctgca cgcagccatg
     6601 gcggacacat tcctggagca catgtgccgg ctggacatcg actccgcgcc catcacggcc
     6661 cgcaacacgg gcatcatctg taccatcggc cctgcctccc gatcggtgga gacgctgaag
     6721 gagatgatca agtctgggat gaacgtggcg cgcatgaact tctcccacgg aactcacgag
     6781 taccatgcgg agaccatcaa gaacgtgcgc acagccacgg aaagctttgc ctcagacccc
     6841 atcctctac
//
