LOCUS       sequence-437371-        7136 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible expression of Zymomonas mobilis pyruvate kinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7136)
  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 7136)
  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: pET15-ZmPYK
FEATURES             Location/Qualifiers
     source          1..7136
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1015..1034
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1054..1131)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1337..1356
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1440..1459
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1440..1458
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1459..1483
                     /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             1498..1520
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1539..1556
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1569..1589
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3076..3094)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3090..3137
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3312..3317,3318..3334,3335..3340))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3367..3385)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3453..3557
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3558..3626,3627..4418)
                     /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(3776..3795)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4589..5177
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5078..5097
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5331..5348
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5363..5502
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5428..5447)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5588..5610
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5604..5795)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7107..7136,1..1053))
                     /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"
ORIGIN
        1 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
       61 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
      121 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
      181 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
      241 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
      301 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
      361 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
      421 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
      481 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
      541 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
      601 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
      661 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
      721 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
      781 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
      841 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
      901 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
      961 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     1021 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     1081 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     1141 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     1201 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     1261 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     1321 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     1381 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt
     1441 aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt
     1501 gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat catcacagca
     1561 gcggcagaga aaacttgtat ttccagggcc atatgaccga gggtctgttc ccgcgtggcc
     1621 gtaaagtgcg tgtggttagc accctgggtc cggcgagcag caccgcggaa cagatccgtg
     1681 accgtttcct ggcgggtgcg gatgtttttc gtattaacat gagccacggc acccacgacg
     1741 agaagaaagt gatcgttgat aacattcgtg cgctggagaa agaattcaac cgtccgacca
     1801 ccatcctgtt tgacctgcag ggcccgaagc tgcgtgttgg tgatttcaaa gagggtaaag
     1861 tgcagctgaa agaaggtcaa accttcacct ttgaccaaga tccgaccctg ggcgacgaga
     1921 cccgtgtgaa cctgccgcac ccggaaattt ttaaggcgct ggataaaggt caccgtctgc
     1981 tgctggacga tggcaagatc gtggttcgtt gcgttgagag cagcccgacc aaaattgtga
     2041 cccgtgtgga agttccgggt ccgctgagcg accacaaggg ctttaacgtt ccggatgttg
     2101 ttattccgct ggcggcgctg accccgaagg atcgtaaaga cctggatttc gcgctgcagg
     2161 agaaagcgga ctgggtggcg ctgagctttg tgcaacgtgt tgaagatgtg atcgaggcga
     2221 aggaactgat taaaggtcgt gcgccgctgc tggttaagct ggagaaaccg gcggcgatcg
     2281 agaacctgga aagcattctg gcggcgaccg atgcggtgat ggttgcgcgt ggtgatctgg
     2341 gcgttgaatg cctgccggaa agcgtgccgc cgacccagaa gcgtatcgtg gagcgtagcc
     2401 gtcaactggg taaaccggtg gttgtggcga ccgcgatgct ggaaagcatg atcaaagcgc
     2461 cggcgccgac ccgtgcggaa gtgagcgacg tggcgaacgc gatctacgag ggtgcggatg
     2521 gcattatgct gagcgcggag agcgcggcgg gtgattggcc gcatgaagcg gttaacatga
     2581 tgcaccgtat cgcgagctac gtggagaacg cgccgggcta tattgaacgt gttcgtttta
     2641 ccccgacccc ggcggaaccg accaccgtgg atgcgctggc ggagaacgcg agcaagaccg
     2701 cggaaaccgt tggtgcgaaa gcgatcattg tgtttaccga gaccggcaaa accgcgcaac
     2761 gtgttagccg tgcgcgtccg gtggcgccga tcctgagcct gaccccggat gcggaagttg
     2821 cgcgtcgtct gggtctggtg tggggcgcgc agccggtgca agttagcacc gttaagaccc
     2881 tggacgaggc gaagaaactg gcggcggaaa ccgcgaagaa atatggtttc gcgaaggcgg
     2941 gcgataaact ggttgtggtt gcgggcgagc cgtttggtaa agcgggcacc accaacatcg
     3001 tggacgttat tgaagcgtaa ctcgaggatc cggctgctaa caaagcccga aaggaagctg
     3061 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     3121 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg atatcccgca agaggcccgg
     3181 cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc cgaggatgac
     3241 gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat ttaactgtga
     3301 taaactaccg cattaaagct tatcgatgat aagctgtcaa acatgagaat tcttgaagac
     3361 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     3421 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     3481 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3541 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     3601 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     3661 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     3721 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     3781 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     3841 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     3901 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     3961 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     4021 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     4081 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     4141 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     4201 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     4261 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     4321 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     4381 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     4441 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     4501 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4561 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4621 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4681 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     4741 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     4801 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     4861 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4921 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4981 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5041 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5101 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5161 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5221 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     5281 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     5341 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     5401 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     5461 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     5521 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     5581 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     5641 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     5701 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     5761 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     5821 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     5881 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     5941 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     6001 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     6061 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     6121 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     6181 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     6241 agcacgatca tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg
     6301 ctgctggaga tggcggacgc gatggatatg ttctgccaag ggttggtttg cgcattcaca
     6361 gttctccgca agaattgatt ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc
     6421 cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga
     6481 ggcagacaag gtatagggcg gcgcctacaa tccatgccaa cccgttccat gtgctcgccg
     6541 aggcggcata aatcgccgtg acgatcagcg gtccaatgat cgaagttagg ctggtaagag
     6601 ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg
     6661 cctgcaacgc gggcatcccg atgccgccgg aagcgagaag aatcataatg gggaaggcca
     6721 tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg
     6781 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     6841 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     6901 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     6961 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     7021 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     7081 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctg
//
