LOCUS       sequence-275437-        8303 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GK5 (isoform 2)-D30N/D280N/D443N mutant in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8303)
  AUTHORS   Zhang D, Tomisato W, Su L, Sun L, Choi JH, Zhang Z, Wang KW, Zhan X,
            Choi M, Li X, Tang M, Castro-Perez JM, Hildebrand S, Murray AR, 
            Moresco EMY, Beutler B
  TITLE     Skin-specific regulation of SREBP processing and lipid biosynthesis 
            by glycerol kinase 5.
  JOURNAL   Proc Natl Acad Sci U S A. 2017 Jun 27;114(26):E5197-E5206. doi: 
            10.1073/pnas.1705312114. Epub 2017 Jun 12.
  PUBMED    28607088
REFERENCE   2  (bases 1 to 8303)
  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"; doi: 
            "10.1073/pnas.1705312114"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2017-06-27- 27"; volume: "114"; issue: "26"; pages: 
            "E5197-E5206"
COMMENT     Sequence Label: pMBP-parallel1-GK5_v2 (D30N+D280N+D443N)
FEATURES             Location/Qualifiers
     source          1..8303
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..80
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             81..1163
                     /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     complement(100..119)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1406..1411,1412..1427,1428..1434)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1442..1458
                     /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     1447..1469
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1528..2628
                     /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="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     2602..2625
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2689..2709
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(4396..4413)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4396..4412)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4405..4427)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4591..4608)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4761..4847
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4939..4966
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        4985..5076
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5077..5145,5146..5937)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5295..5314)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(5979..6492)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6174..6195)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6386..6405
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      6603..7191
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7092..7111
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7345..7362
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7377..7517
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7443..7462)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7603..7625
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7619..7810)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
       61 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
      121 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
      181 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
      241 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
      301 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
      361 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
      421 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
      481 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
      541 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
      601 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
      661 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
      721 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
      781 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
      841 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg
      901 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
      961 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     1021 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     1081 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     1141 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
     1201 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg
     1261 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
     1321 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
     1381 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga
     1441 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga
     1501 gcacttcacc aacaaggacc atagattatg aaaactgaag aaggtaaact ggtaatctgg
     1561 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat
     1621 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt
     1681 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac
     1741 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat
     1801 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt
     1861 gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa
     1921 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac
     1981 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag
     2041 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg
     2101 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac
     2161 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg
     2221 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc
     2281 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt
     2341 ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg
     2401 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag
     2461 ttggcgaaag atccacgtat tgccgccaca atggaaaacg cccagaaagg tgaaatcatg
     2521 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc
     2581 gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg
     2641 aacaacaaca acaataacaa taacaacaac ctcgggatcg atacgaccga aaacctgtat
     2701 tttcagggcg ccatggatcc ggaattcatg tcagggcagc aggagcgtgc agagcggcag
     2761 cgagaggaac tgagcgcgtc ggcctcgccg ccctcccgct tcgtgctggg gctgaacgtg
     2821 ggcagcacgg tgatccgctg ccacgtctat gaccagacgg cgcgagtgcg aggctccagc
     2881 gcgcagaagg tagaaaatgt ttaccctcaa cctggctggg ttgaaatcga tcctgattct
     2941 ctctgggctc agtttgttgc cgtaataaaa gatgctgtca aagctgcagg ggtgcagatg
     3001 aatcaaattg tcggtcttgg catatcaaca cagagagcca ctttcattac atggaacaag
     3061 aaaacaggcc atcattttca caacttcatt agctggcaag acctaagggc agctgaactt
     3121 gtaaagtcct ggaataattc tctcataatg aagctattgc atggggccac ccgagtcctt
     3181 catttcttca gtagaagtaa agtaatgcta acggtcagcc gcttcaattt cagcacccag
     3241 catgccacct taagattgac ctggatttta caaaacctat ctgaggtgaa aagggcagtt
     3301 gaagaagaca attgttgctt tgggacgatt gatacctggc tgttatacaa gctcacgaaa
     3361 ggttcttcct acgccacgga ttattcaaat gccagcacca ctggattttt tgatccctat
     3421 gcgatgcgct ggagcaggct cataaccacc atggtctcca tcccactgtc tatcctgcct
     3481 cctgtgaagg ataccagcta taattttgga tcagtggatg agaagatatt tggcgtgcct
     3541 ataccagttg ttgccttggt tggtaaccag cagtcggcca tgtttggaga gtgctgcttc
     3601 gagacgggag atgtgaagct aaccatggga actggaacat ttttggatat caatactgga
     3661 aaaaaccttc agcatgttaa tggaggcttt tatccactaa ttggatggaa gattgggcaa
     3721 gaacttgtat gtttagctga aggcaatgca ggagacacgg gcaccgccat aatgtgggct
     3781 caaaaattag atctcttcac agacgctgct gagactgaaa aaatggccct gagcctggaa
     3841 gattctgaag gggtttactt tgttccgtcg tttagtggat tacaggctcc attaaatgac
     3901 ccctgtgcct gtgcctcttt tatgggtttg aagcactcca ctaataaata ccatcttgtc
     3961 cgagcaatat tggagtcaat agctttcagg aacaaacagt tgtacgacat gctgcagaga
     4021 gaaatccaga ttcccgtcac gaacatccgg gcaaatggag gcgtgtgtaa caacgctttc
     4081 gtcatgcaga tgacttcaga cctgataaat gagaagatag acagacctgc ccacttcgac
     4141 atgtcctgct tgggggcagc ctctctggct ggcctcgctg ttggattttg ggcggacaag
     4201 gaagagctcc agaagctgag gcaaagcgag atggttttca agcctcagaa gaagtggcag
     4261 gaatacgagg tgaatatgga aaactgggta aaagcggtga agcgctccat gaattggtat
     4321 aacaagacgt agactagtgc ggccgctttc gaatctagag cctgcagtct cgaggcatgc
     4381 ggtaccaagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     4441 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     4501 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcagcttgg
     4561 ctgttttggc ggatgagata agattttcag cctgatacag attaaatcag aacgcagaag
     4621 cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat
     4681 gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc cccatgcgag
     4741 agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
     4801 gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg
     4861 atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg
     4921 ccaggcatca aattaagcag aaggccatcc tgacggatgg cctttttgcg tttctacaaa
     4981 ctcttttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5041 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5101 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5161 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5221 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgtttccc aatgatgagc
     5281 acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa
     5341 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     5401 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     5461 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5521 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     5581 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     5641 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     5701 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     5761 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     5821 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     5881 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     5941 tcagaccaag tttactcata tatactttag attgatttac cccggttgat aatcagaaaa
     6001 gccccaaaaa caggaagatt gtataagcaa atatttaaat tgtaaacgtt aatattttgt
     6061 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     6121 gcaaaatccc ttataaatca aaagaatagc ccgagatagg gttgagtgtt gttccagttt
     6181 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     6241 atcagggcga tggcccacta cgtgaaccat cacccaaatc aagttttttg gggtcgaggt
     6301 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     6361 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     6421 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     6481 tacagggcgc gtaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6541 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6601 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6661 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6721 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     6781 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6841 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6901 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6961 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     7021 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     7081 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     7141 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     7201 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     7261 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     7321 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     7381 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc
     7441 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     7501 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     7561 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     7621 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     7681 ggtcgtgcag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     7741 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     7801 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     7861 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     7921 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     7981 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     8041 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     8101 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     8161 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     8221 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtggcc
     8281 aggacccaac gctgcccgaa att
//
