LOCUS       sequence-465789-        6413 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For bacterial expression of Flag-tagged E. coli LplA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6413)
  AUTHORS   Liu S, Kormos BL, Knafels JD, Sahasrabudhe PV, Rosado A, Sommese RF,
            Reyes AR, Ward J, Roth Flach RJ, Wang X, Buzon LM, Reese MR, 
            Bhattacharya SK, Omoto K, Filipski KJ
  TITLE     Structural studies identify angiotensin II receptor blocker-like 
            compounds as branched-chain ketoacid dehydrogenase kinase 
            inhibitors.
  JOURNAL   J Biol Chem. 2023 Mar;299(3):102959. doi: 10.1016/j.jbc.2023.102959.
            Epub 2023 Jan 28.
  PUBMED    36717078
REFERENCE   2  (bases 1 to 6413)
  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: "J Biol 
            Chem."; date: "2023-03"; volume: "299(3):102959. doi"; pages: " 
            10.1016/j.jbc.2023.102959. Epub 2023 Jan 28"
COMMENT     Sequence Label: pETDuet-1 Flag-LplA
FEATURES             Location/Qualifiers
     source          1..6413
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        214..232
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    233..257
                     /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             303..326
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1362..1406
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1444..1462)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1458..1505
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1542..1997
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1629..1648)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1839..1860
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2112..2903,2904..2972))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2735..2754
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2973..3065)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3146..3734
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3635..3654
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3888..3905
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3920..4059
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3985..4004)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4145..4167
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4161..4352)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4924..6006)
                     /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     5968..5987
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6007..6084)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6290..6309
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(6397..6413,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
      121 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
      181 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
      241 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
      301 tggattacaa agatgatgat gataagatga gcaccctgcg tctgctgatt agcgacagct
      361 atgacccgtg gttcaacctg gcggttgagg aatgcatctt ccgtcagatg ccggcgaccc
      421 aacgtgtgct gtttctgtgg cgtaacgcgg acaccgtggt tattggccgt gcgcagaacc
      481 cgtggaaaga atgcaacacc cgtcgtatgg aggaagataa cgttcgtctg gcgcgtcgta
      541 gcagcggtgg cggtgcggtt ttccacgacc tgggcaacac ctgcttcacc tttatggcgg
      601 gtaaaccgga atacgataag accatcagca ccagcattgt gctgaacgcg ctgaacgcgc
      661 tgggcgttag cgcggaggcg agcggtcgta acgacctggt ggttaaaacc gtggaaggcg
      721 atcgtaaggt tagcggtagc gcgtaccgtg aaaccaaaga ccgtggcttt caccacggca
      781 ccctgctgct gaacgcggac ctgagccgtc tggcgaacta tctgaacccg gataagaaaa
      841 agctggcggc gaagggtatc accagcgtgc gtagccgtgt taccaacctg accgaactgc
      901 tgccgggtat cacccacgag caagtgtgcg aagcgattac cgaggcgttc tttgcgcact
      961 acggcgagcg tgttgaggcg gaaatcatta gcccgaacaa aaccccggac ctgccgaact
     1021 tcgcggaaac ctttgcgcgt cagagcagct gggagtggaa cttcggccaa gcgccggcgt
     1081 ttagccacct gctggacgaa cgtttcacct ggggcggtgt ggaactgcac tttgatgttg
     1141 agaagggtca cattacccgt gcgcaggtgt tcaccgatag cctgaacccg gcgccgctgg
     1201 aggcgctggc gggccgtctg caaggttgcc tgtatcgtgc ggacatgctg cagcaagagt
     1261 gcgaagcgct gctggttgat tttccggagc aagagaaaga actgcgtgaa ctgagcgcgt
     1321 ggatggcggg tgcggtgcgt taaggtaccc tcgagtctgg taaagaaacc gctgctgcga
     1381 aatttgaacg ccagcacatg gactcgtcta ctagcgcagc ttaattaacc taggctgctg
     1441 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     1501 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
     1561 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     1621 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     1681 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     1741 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     1801 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     1861 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     1921 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     1981 attaacgttt acaatttctg gcggcacgat ggcatgagat tatcaaaaag gatcttcacc
     2041 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     2101 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     2161 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     2221 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     2281 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     2341 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     2401 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     2461 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     2521 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     2581 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     2641 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     2701 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     2761 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     2821 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     2881 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     2941 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaatc atgattgaag
     3001 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     3061 acaaataggt catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     3121 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3181 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3241 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     3301 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3361 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3421 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3481 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3541 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3601 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3661 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3721 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3781 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3841 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3901 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3961 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     4021 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     4081 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4141 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     4201 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     4261 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     4321 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     4381 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     4441 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     4501 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     4561 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     4621 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     4681 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     4741 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     4801 acgatcatgc tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     4861 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     4921 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4981 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     5041 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     5101 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     5161 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg caccaacgcg
     5221 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     5281 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     5341 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     5401 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     5461 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     5521 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     5581 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     5641 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     5701 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     5761 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     5821 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     5881 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     5941 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     6001 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     6061 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     6121 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     6181 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     6241 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     6301 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     6361 cgtagaggat cgagatcgat ctcgatcccg cgaaattaat acgactcact ata
//
