LOCUS       sequence-190601-        6684 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses His-tagged mouse ECHB (35-475) in E.coli cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6684)
  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 6684)
  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-ECHB(35-475)
FEATURES             Location/Qualifiers
     source          1..6684
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      26..614
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     515..534
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     768..785
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    800..939
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(865..884)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1025..1047
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1041..1232)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2041..3123)
                     /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     3085..3104
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3124..3201)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3407..3426
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3510..3529
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3510..3528
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3529..3553
                     /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             3568..3590
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3609..3626
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3636..3653
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3657..3689
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             5058..5075
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5128..5146)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5142..5189
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5226..5681
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5313..5332)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5523..5544
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5773..6588)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5773..6588)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6496..6515
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6580..6599
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
       61 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
      121 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
      181 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
      241 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
      301 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
      361 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
      421 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
      481 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
      541 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
      601 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
      661 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
      721 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
      781 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
      841 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
      901 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
      961 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     1021 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     1081 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     1141 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     1201 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     1261 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     1321 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     1381 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     1441 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     1501 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     1561 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     1621 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     1681 acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     1741 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     1801 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     1861 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     1921 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     1981 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     2041 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     2101 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     2161 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     2221 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     2281 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
     2341 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     2401 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     2461 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     2521 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     2581 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     2641 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     2701 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     2761 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     2821 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     2881 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     2941 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     3001 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     3061 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     3121 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     3181 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     3241 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     3301 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     3361 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     3421 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     3481 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
     3541 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg
     3601 gcagcagcca tcatcatcat catcacagca gcggcctggt gccgcgcggc agccatatgg
     3661 ctagcatgac tggtggacag caaatgggtc gcggatccga attctctgcc ccagctgtcc
     3721 agaccaagtc aaagaagact ttagcaaaac ccaatatgaa gaatattgtg gtggtggaag
     3781 gggtccgcat tccatttctg ctgtcaggca cttcgtataa agacctaatg ccacatgact
     3841 tggctagagc tgcactttcg ggtttgttgc atcggaccaa tattccaaag gatgttgttg
     3901 attatatcat ctttggtaca gttattcagg aagtgaaaac aagcaatgtg gctagagagg
     3961 ctgccctggg agctggcttc tctgataaga ctccagctca cactgtcacc atggcttgta
     4021 tctcttcaaa ccaagccatg accacagctg ttggtctgat agcttctggc cagtgtgatg
     4081 tcgtcgtggc tggtggtgtt gagttaatgt ctgatgtccc tattcgtcat tcaagaaata
     4141 tgaggaaaat gatgcttgat ctcaataaag ccaagactct gggccagcgc ctgtccttac
     4201 tcagtaaatt cagattgaat tttctgtccc ctgagctccc tgcagtggct gagttctcca
     4261 ctaatgagac catgggccac tctgcagacc gactggctgc tgcctttgct gtttctcgaa
     4321 tggaacagga tgaatatgca ctgcgttctc atagtctggc caagaaggca caggatgaag
     4381 gacacctttc tgatattgta cccttcaaag taccaggaaa ggacacagtt accaaagata
     4441 atgggatccg tccttcctca ctggagcaaa tggccaaact aaaacctgcg ttcatcaaac
     4501 cctatggcac agtgacagct gcaaattctt ctttcctgac tgatggcgct tctgcgatgc
     4561 taatcatgtc agaggacaga gctctggcca tgggttataa accaaaggca tatttgaggg
     4621 attttatata tgtgtcccag gatccgaaag atcagctttt actcggacca acatatgcta
     4681 ctccaaaagt tttagaaaag gcagggttaa ccatgaatga tatcgatgct tttgaatttc
     4741 atgaagcctt ctcaggccag attttagcta actttaaagc catggattct gattggtttg
     4801 cacaaaacta catgggtagg aaaaccaagg ttggatcacc tcctctggag aagtttaata
     4861 tctggggcgg atcactgtct ctggggcacc cttttggagc cactggctgt cggctggtca
     4921 tggcagctgc caacagactg aggaaggatg gaggccagta tgctttagtg gctgcctgtg
     4981 cagctggagg acagggtcat gctatgattg tggaagctta ccccaaatga gtcgacaagc
     5041 ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
     5101 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     5161 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     5221 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     5281 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     5341 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     5401 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     5461 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     5521 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     5581 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     5641 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     5701 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     5761 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     5821 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     5881 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     5941 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     6001 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     6061 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     6121 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     6181 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     6241 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     6301 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     6361 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     6421 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     6481 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     6541 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     6601 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt
     6661 tttcgttcca ctgagcgtca gacc
//
