LOCUS       sequence-129101-        6974 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of G2K (d-glycerate 2-kinase).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6974)
  AUTHORS   Sims, P, Reed, G
  TITLE     Method for the enzymatic synthesis of 2-phospho-d-glycerate from 
            adenosine 5′-triphosphate and d-glycerate via d-glycerate-2-kinase
  JOURNAL   Journal of Molecular Catalysis B: Enzymatic 32 (2005) 77–81
REFERENCE   2  (bases 1 to 6974)
  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: "Journal of 
            Molecular Catalysis B: Enzymatic 32 (2005) 77–81"
COMMENT     Sequence Label: pET-G2K
FEATURES             Location/Qualifiers
     source          1..6974
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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             1554..1595
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1605..1622
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1679..1697)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1693..1740
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1915..1920,1921..1937,1938..1943))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1974..1992)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2060..2164
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2165..2233,2234..3025)
                     /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(2383..2402)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3196..3784
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3685..3704
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3938..3955
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3970..4109
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4035..4054)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4195..4217
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4211..4402)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5714..6796)
                     /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     6758..6777
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6797..6874)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggaatt caggagccct
      301 tcaccatggc gtattgcaat ccgggcctgg aatccaggcc gaataagaga aacgccctcc
      361 ggcgtcatgt ggtaacaggc ataggtatga aaatcgtaat cgccccagac tcttataaag
      421 aaagtttatc tgccagcgag gttgcgcagg cgatagaaaa aggatttcgg gaaatttttc
      481 ctgatgcaca gtacgtttct gttccggttg ccgacggtgg cgaaggaacg gtggaagcga
      541 tgattgcagc cacccagggg gctgaacgtc acgcctgggt tacagggccg ctgggcgaga
      601 aagtgaatgc cagttggggg atctccggcg atggcaaaac cgcgtttatt gaaatggcgg
      661 cggccagtgg gctggagctg gtacctgcgg aaaaacgcga tccactcgtg accacttcac
      721 gcggcacagg cgagttaatc ctgcaggcgc tggagagcgg tgcgacaaac attattatcg
      781 gcattggcgg cagcgctaca aatgatggcg gcgcaggcat ggtacaggcg ctgggggcga
      841 aattatgcga cgccaacggc aatgaaattg gttttggcgg cggtagtctt aatactctga
      901 atgatattga tatttccggc ctcgatccgc gcttaaaaga ttgcgtcatt cgcgtcgctt
      961 gtgatgtcac caatccgctg gtgggcgata acggcgcatc gcgcatcttt ggcccacaaa
     1021 agggagccag tgaagcgatg attgttgagc tggacaataa cctctctcac tatgccgagg
     1081 tcattaaaaa agcgctgcat gttgatgtga aagatgtccc cggtgcagga gctgcgggtg
     1141 gtatgggcgc ggcgctaatg gcgtttcttg gtgcggaact gaaaagtggt attgaaatcg
     1201 tcactacggc gctgaatctg gaggaacata ttcacgattg tacgctggtg atcaccggtg
     1261 aagggcgtat tgacagccag agtattcacg ggaaggtacc gattggtgtc gcaaacgtgg
     1321 cgaagaagta ccataaaccg gtgattggca ttgcgggtag cctgaccgat gatgttggcg
     1381 ttgtacatca gcatggcatt gatgcggtct tcagcgtatt gaccagcata ggtacgttgg
     1441 acgaagcatt ccgcggggct tatgacaata tctgccgtgc ttcacgtaat atcgccgcga
     1501 cactggcgat tggaatgcgc aacgcgaagg gcgagctcaa ttcgaagctt gaaggtaagc
     1561 ctatccctaa ccctctcctc ggtctcgatt ctacgcgtac cggtcatcat caccatcacc
     1621 attgagtttg atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     1681 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     1741 ctgaaaggag gaactatatc cggatatccc gcaagaggcc cggcagtacc ggcataacca
     1801 agcctatgcc tacagcatcc agggtgacgg tgccgaggat gacgatgagc gcattgttag
     1861 atttcataca cggtgcctga ctgcgttagc aatttaactg tgataaacta ccgcattaaa
     1921 gcttatcgat gataagctgt caaacatgag aattaattct tgaagacgaa agggcctcgt
     1981 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     2041 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2101 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     2161 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     2221 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     2281 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     2341 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     2401 atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     2461 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     2521 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     2581 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     2641 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     2701 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     2761 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     2821 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     2881 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     2941 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     3001 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     3061 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     3121 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     3181 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     3241 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     3301 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     3361 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     3421 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3481 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3541 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3601 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3661 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3721 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3781 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3841 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3901 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3961 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     4021 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     4081 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     4141 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4201 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     4261 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     4321 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     4381 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     4441 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     4501 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     4561 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     4621 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     4681 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     4741 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     4801 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     4861 gcacccgtgg ccaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg
     4921 cggacgcgat ggatatgttc tgccaagggt tggtttgcgc attcacagtt ctccgcaaga
     4981 attgattggc tccaattctt ggagtggtga atccgttagc gaggtgccgc cggcttccat
     5041 tcaggtcgag gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta
     5101 tagggcggcg cctacaatcc atgccaaccc gttccatgtg ctcgccgagg cggcataaat
     5161 cgccgtgacg atcagcggtc caatgatcga agttaggctg gtaagagccg cgagcgatcc
     5221 ttgaagctgt ccctgatggt cgtcatctac ctgcctggac agcatggcct gcaacgcggg
     5281 catcccgatg ccgccggaag cgagaagaat cataatgggg aaggccatcc agcctcgcgt
     5341 cgcgaacgcc agcaagacgt agcccagcgc gtcggccgcc atgccggcga taatggcctg
     5401 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     5461 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     5521 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     5581 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     5641 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     5701 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5761 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     5821 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     5881 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     5941 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
     6001 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     6061 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     6121 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     6181 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     6241 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     6301 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     6361 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     6421 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     6481 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     6541 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     6601 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     6661 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     6721 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     6781 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     6841 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     6901 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     6961 aggaatggtg catg
//
