LOCUS       sequence-189215-        7574 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses flag-tagged mouse ECHA (37-768) 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)-FLAG (no His-tag)
FEATURES             Location/Qualifiers
     source          1..7574
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     956..975
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(995..1072)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1278..1297
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1385..1403
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1404..1428
                     /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             1443..1465
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1484..1501
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        1615..1633
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1634..1658
                     /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             3885..3908
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3924..3968
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4006..4024)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4020..4067
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4104..4559
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4191..4210)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4401..4422
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4674..5465,5466..5534))
                     /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     5297..5316
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5535..5627)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5708..6296
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6197..6216
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6450..6467
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6482..6621
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6547..6566)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6707..6729
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6723..6914)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7486..7574,1..994))
                     /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 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
       61 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
      121 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
      181 ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
      241 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
      301 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
      361 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
      421 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
      481 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
      541 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
      601 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
      661 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
      721 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
      781 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
      841 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
      901 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
      961 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     1021 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     1081 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     1141 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     1201 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     1261 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     1321 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatcgatct cgatcccgcg
     1381 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     1441 attttgttta actttaagaa ggagatatac catgggcagc agccatcacc atcatcacca
     1501 cagccaggat ccgaattcga gctcggcgcg cctgcaggtc gacaagcttg cggccgcata
     1561 atgcttaagt cgaacagaaa gtaatcgtat tgtacacggc cgcataatcg aaattaatac
     1621 gactcactat aggggaattg tgagcggata acaattcccc atcttagtat attagttaag
     1681 tataagaagg agatatacat atgaccagaa cccatattaa ttatggcgtc aaaggggatg
     1741 tggcagttat tcggattaac tcacccaatt caaaggtaaa tacattgaat aaagaagtac
     1801 agtcagagtt catagaagtc atgaacgaaa tctgggccaa cgaccaaatc aggagtgctg
     1861 tccttatctc atcaaagcct ggctgctttg tcgcaggggc tgacatcaac atgctatcct
     1921 cttgtacaac cccccaagaa gcaacacgaa tatcacagga aggtcagaga atgtttgaga
     1981 aacttgaaaa gtccccaaag cccgttgtgg ccgccatcag tggatcctgc ttgggaggag
     2041 gacttgagct tgccatagca tgccaataca gaatagcaac aaaagacaga aaaacagtat
     2101 taggtgtccc tgaagtgttg ctggggatct taccaggagc cggaggtacc cagagactgc
     2161 ccaaaatggt gggtgtgcct gctgcatttg acatgatgct gactggcagg aacattcgtg
     2221 cagacagagc aaagaaaatg ggactggttg accagttggt ggaaccgctg ggaccaggaa
     2281 taaaatcacc agaagaaagg acaattgaat acctagagga agttgcagtt aattttgcca
     2341 aaggcctggc tgatcggaaa gtctctgcaa agcagagcaa aggccttgtg gaaaagctga
     2401 caacgtatgc catgactgtc ccatttgtca gacaacaggt ttacaaaaca gttgaagaaa
     2461 aagtgaagaa gcagactaaa ggcctttatc ctgccccttt gaaaataatt gatgctgtga
     2521 aggctggact tgagcaaggc agtgacgctg gttatcttgc tgaatcgcag aaatttggag
     2581 agcttgcatt gaccaaagaa tcgaaggccc tgatggggct ttataacggc caggtcctgt
     2641 gcaagaaaaa taaatttgga gcaccacaga agaacgttca gcagctagcc atccttggcg
     2701 cagggctgat gggggctggc attgcccagg tctctgtgga taagggactg aaaactcttc
     2761 ttaaagacac cacagtgacg gggctgggcc ggggacagca gcaagtgttc aaagggctga
     2821 acgacaaagt gaagaagaaa gctctcacat catttgaaag ggattccatc ttcagcaact
     2881 tgattgggca gctggattac aaaggctttg agaaggccga catggtgatt gaggctgtgt
     2941 ttgaggacct cggtgtaaag cacaaagtgt taaaggaagt ggaaagcgtg actccagaac
     3001 actgtatctt tgccagcaac acatctgctc tcccaatcaa tcaaattgct gctgtcagca
     3061 aaaggcctga gaaggtgatc ggcatgcact acttctcccc cgtggacaag atgcagctcc
     3121 tagaaatcat caccactgac aaaacctcca aggacacaac ggcctctgcc gtggccgtgg
     3181 ggctcaggca agggaaggtc atcattgtgg tcaaggacgg acccggcttc tacaccacca
     3241 ggtgtcttgc acccatgatg tctgaagtca tgcgaatcct ccaggaagga gttgacccta
     3301 agaagctgga cgccttgacc acaggtttcg gcttccctgt gggtgctgcc acgctggcag
     3361 acgaagtggg tgtggatgta gcacagcacg tagcagaaga tctaggcaaa gcctttgggg
     3421 agcgatttgg aggtggcagc gtagaactgc tgaaacagat ggtctccaag ggcttcttag
     3481 gtcgcaagtc tgggaagggc ttttacatct atcaggaggg ctcaaagaat aagagtttga
     3541 attctgaaat ggataatatc ttggcaaatc tgaggctgcc tgccaagccc gaggtctcct
     3601 ctgatgaaga cgtccaatac cgtgtgataa caaggttcgt gaacgaggca gtcctgtgcc
     3661 tacaggaagg gatcctggcc acaccggcag agggagacat cggagctgtc tttgggcttg
     3721 gctttccccc ttgtctcgga gggcccttcc gctttgtgga tctgtatggt gctcagaagg
     3781 tagtggaccg gctacggaag tacgagtctg cctatggaac acagtttacc ccatgccagc
     3841 tgctcctcga ccacgctaac aactctagca agaagttcta ccaggattac aaggatgacg
     3901 acgataagtg actcgagtct ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca
     3961 tggactcgtc tactagcgca gcttaattaa cctaggctgc tgccaccgct gagcaataac
     4021 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     4081 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4141 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     4201 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4261 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     4321 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4381 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     4441 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     4501 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc
     4561 tggcggcacg atggcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4621 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     4681 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     4741 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     4801 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     4861 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     4921 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     4981 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5041 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5101 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5161 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5221 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5281 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     5341 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5401 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5461 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5521 tgttgaatac tcatactctt cctttttcaa tcatgattga agcatttatc agggttattg
     5581 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gtcatgacca
     5641 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     5701 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     5761 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     5821 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     5881 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     5941 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     6001 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6061 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6121 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6181 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6241 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     6301 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     6361 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     6421 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     6481 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     6541 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     6601 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     6661 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     6721 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     6781 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     6841 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     6901 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     6961 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     7021 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     7081 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     7141 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     7201 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     7261 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     7321 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gctagtcatg
     7381 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     7441 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     7501 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     7561 ggtttgcgta ttgg
//
