LOCUS       sequence-190596-        7574 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses-flag tagged mouse ECHA(37-768) K351R in E.coli cells (no 
            His-tag).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7574)
  AUTHORS   Sadhukhan S, Liu X, Ryu D, Nelson OD, Stupinski JA, Li Z, Chen W, 
            Zhang S, Weiss RS, Locasale JW, Auwerx J, Lin H
  TITLE     Metabolomics-assisted proteomics identifies succinylation and SIRT5 
            as important regulators of cardiac function.
  JOURNAL   Proc Natl Acad Sci U S A. 2016 Apr 19;113(16):4320-5. doi: 
            10.1073/pnas.1519858113. Epub 2016 Apr 5.
  PUBMED    27051063
REFERENCE   2  (bases 1 to 7574)
  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.1519858113"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2016-04-19- 19"; volume: "113"; issue: "16"; pages: "4320-5"
COMMENT     Sequence Label: pET-Duet-ECHA(37-763) K351R-FLAG (no His-tag)
FEATURES             Location/Qualifiers
     source          1..7574
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      13..601
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     502..521
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     755..772
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    787..926
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(852..871)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1012..1034
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1028..1219)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1791..2873)
                     /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     2835..2854
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2874..2951)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3157..3176
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3264..3282
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3283..3307
                     /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             3322..3344
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3363..3380
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        3494..3512
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3513..3537
                     /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             5764..5787
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5803..5847
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(5885..5903)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5899..5946
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5983..6438
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6070..6089)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6280..6301
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(6553..7344,7345..7413))
                     /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     7176..7195
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7414..7506)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
       61 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
      121 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
      181 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
      241 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
      301 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
      361 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
      421 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
      481 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
      541 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
      601 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
      661 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
      721 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
      781 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
      841 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
      901 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
      961 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     1021 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     1081 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     1141 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     1201 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     1261 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     1321 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     1381 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     1441 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     1501 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     1561 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     1621 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgctag
     1681 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     1741 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     1801 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     1861 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     1921 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     1981 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     2041 tcttcggtat cgtcgtatcc cactaccgag atgtccgcac caacgcgcag cccggactcg
     2101 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     2161 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     2221 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     2281 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     2341 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     2401 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     2461 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     2521 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     2581 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     2641 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     2701 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     2761 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     2821 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     2881 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     2941 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     3001 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
     3061 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
     3121 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
     3181 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
     3241 gatcgatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa
     3301 caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatatacc atgggcagca
     3361 gccatcacca tcatcaccac agccaggatc cgaattcgag ctcggcgcgc ctgcaggtcg
     3421 acaagcttgc ggccgcataa tgcttaagtc gaacagaaag taatcgtatt gtacacggcc
     3481 gcataatcga aattaatacg actcactata ggggaattgt gagcggataa caattcccca
     3541 tcttagtata ttagttaagt ataagaagga gatatacata tgaccagaac ccatattaat
     3601 tatggcgtca aaggggatgt ggcagttatt cggattaact cacccaattc aaaggtaaat
     3661 acattgaata aagaagtaca gtcagagttc atagaagtca tgaacgaaat ctgggccaac
     3721 gaccaaatca ggagtgctgt ccttatctca tcaaagcctg gctgctttgt cgcaggggct
     3781 gacatcaaca tgctatcctc ttgtacaacc ccccaagaag caacacgaat atcacaggaa
     3841 ggtcagagaa tgtttgagaa acttgaaaag tccccaaagc ccgttgtggc cgccatcagt
     3901 ggatcctgct tgggaggagg acttgagctt gccatagcat gccaatacag aatagcaaca
     3961 aaagacagaa aaacagtatt aggtgtccct gaagtgttgc tggggatctt accaggagcc
     4021 ggaggtaccc agagactgcc caaaatggtg ggtgtgcctg ctgcatttga catgatgctg
     4081 actggcagga acattcgtgc agacagagca aagaaaatgg gactggttga ccagttggtg
     4141 gaaccgctgg gaccaggaat aaaatcacca gaagaaagga caattgaata cctagaggaa
     4201 gttgcagtta attttgccaa aggcctggct gatcggaaag tctctgcaaa gcagagcaaa
     4261 ggccttgtgg aaaagctgac aacgtatgcc atgactgtcc catttgtcag acaacaggtt
     4321 tacaaaacag ttgaagaaaa agtgaagaag cagactaaag gcctttatcc tgcccctttg
     4381 aaaataattg atgctgtgaa ggctggactt gagcaaggca gtgacgctgg ttatcttgct
     4441 gaatcgcaga aatttggaga gcttgcattg accaaagaat cgaaggccct gatggggctt
     4501 tataacggcc aggtcctgtg caagaggaat aaatttggag caccacagaa gaacgttcag
     4561 cagctagcca tccttggcgc agggctgatg ggggctggca ttgcccaggt ctctgtggat
     4621 aagggactga aaactcttct taaagacacc acagtgacgg ggctgggccg gggacagcag
     4681 caagtgttca aagggctgaa cgacaaagtg aagaagaaag ctctcacatc atttgaaagg
     4741 gattccatct tcagcaactt gattgggcag ctggattaca aaggctttga gaaggccgac
     4801 atggtgattg aggctgtgtt tgaggacctc ggtgtaaagc acaaagtgtt aaaggaagtg
     4861 gaaagcgtga ctccagaaca ctgtatcttt gccagcaaca catctgctct cccaatcaat
     4921 caaattgctg ctgtcagcaa aaggcctgag aaggtgatcg gcatgcacta cttctccccc
     4981 gtggacaaga tgcagctcct agaaatcatc accactgaca aaacctccaa ggacacaacg
     5041 gcctctgccg tggccgtggg gctcaggcaa gggaaggtca tcattgtggt caaggacgga
     5101 cccggcttct acaccaccag gtgtcttgca cccatgatgt ctgaagtcat gcgaatcctc
     5161 caggaaggag ttgaccctaa gaagctggac gccttgacca caggtttcgg cttccctgtg
     5221 ggtgctgcca cgctggcaga cgaagtgggt gtggatgtag cacagcacgt agcagaagat
     5281 ctaggcaaag cctttgggga gcgatttgga ggtggcagcg tagaactgct gaaacagatg
     5341 gtctccaagg gcttcttagg tcgcaagtct gggaagggct tttacatcta tcaggagggc
     5401 tcaaagaata agagtttgaa ttctgaaatg gataatatct tggcaaatct gaggctgcct
     5461 gccaagcccg aggtctcctc tgatgaagac gtccaatacc gtgtgataac aaggttcgtg
     5521 aacgaggcag tcctgtgcct acaggaaggg atcctggcca caccggcaga gggagacatc
     5581 ggagctgtct ttgggcttgg ctttccccct tgtctcggag ggcccttccg ctttgtggat
     5641 ctgtatggtg ctcagaaggt agtggaccgg ctacggaagt acgagtctgc ctatggaaca
     5701 cagtttaccc catgccagct gctcctcgac cacgctaaca actctagcaa gaagttctac
     5761 caggattaca aggatgacga cgataagtga ctcgagtctg gtaaagaaac cgctgctgcg
     5821 aaatttgaac gccagcacat ggactcgtct actagcgcag cttaattaac ctaggctgct
     5881 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     5941 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
     6001 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     6061 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     6121 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     6181 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     6241 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     6301 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     6361 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     6421 tattaacgtt tacaatttct ggcggcacga tggcatgaga ttatcaaaaa ggatcttcac
     6481 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6541 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6601 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6661 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6721 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6781 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6841 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6901 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6961 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     7021 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     7081 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     7141 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     7201 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7261 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7321 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7381 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat catgattgaa
     7441 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7501 aacaaatagg tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     7561 cccgtagaaa agat
//
