LOCUS       sequence-218560-        6191 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6191)
  AUTHORS   Shen H, Campanello GC, Flicker D, Grabarek Z, Hu J, Luo C, Banerjee 
            R, Mootha VK
  TITLE     The Human Knockout Gene CLYBL Connects Itaconate to Vitamin B12.
  JOURNAL   Cell. 2017 Nov 2;171(4):771-782.e11. doi: 
            10.1016/j.cell.2017.09.051. Epub 2017 Oct 19.
  PUBMED    29056341
REFERENCE   2  (bases 1 to 6191)
  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: 
            "10.1016/j.cell.2017.09.051"; journalName: "Cell"; date: "2017-11-2-
            2"; volume: "171"; issue: "4"; pages: "771-782.e11"
COMMENT     Sequence Label: HsCLYBL(aa23-340) D320N-His
FEATURES             Location/Qualifiers
     source          1..6191
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     65..84
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        65..83
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    84..108
                     /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             123..145
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1116..1133
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1186..1204)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1200..1247
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1284..1739
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1371..1390)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1581..1602
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1831..2646)
                     /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(1831..2646)
                     /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     2554..2573
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2638..2657
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2768..3356
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3257..3276
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3510..3527
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3542..3681
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3607..3626)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3767..3789
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3783..3974)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4783..5865)
                     /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     5827..5846
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5866..5943)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6149..6168
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatcgatct cgatcccgcg
       61 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
      121 attttgttta actttaagaa ggagatatac atatggccag cttagccgca gatatcccgc
      181 gtctgggcta cagtagcagc agccaccaca agtacatccc tcgtcgcgca gtgctgtatg
      241 tgccgggcaa cgatgagaag aagattaaga agattccgag cctgaacgtg gattgcgcag
      301 tgctggactg tgaggacggc gtggcagcca acaaaaagaa cgaagcccgc ctgcgcatcg
      361 tgaagaccct ggaggacatc gatctgggcc cgaccgagaa atgcgtgcgt gtgaacagcg
      421 ttagcagcgg tctggcagaa gaggatttag agaccctgct gcagagccgc gttctgccga
      481 gcagtctgat gctgccgaag gtggagagcc cggaagagat ccagtggttc gccgataagt
      541 tcagcttcca cctgaagggt cgcaagctgg agcagccgat gaacctgatc cctttcgtgg
      601 agacagccat gggcctgctg aatttcaaag ccgtgtgcga ggagaccctg aaggttggtc
      661 cgcaagtggg cctgttcctg gacgccgttg tgttcggcgg tgaggacttc cgcgcaagca
      721 ttggcgcaac cagcagtaag gagaccctgg atatcctgta tgcccgccag aaaatcgtgg
      781 tgatcgccaa ggccttcggc ctgcaggcca tcgacctggt gtacattgac ttccgtgatg
      841 gcgccggctt actgcgtcag agccgcgaag gtgcagccat gggcttcacc ggtaagcagg
      901 ttatccaccc taaccaaatc gccgtggtgc aggaacagtt tagcccgagc ccggagaaga
      961 tcaagtgggc cgaagagctg atcgccgcct tcaaggagca tcagcagctg ggcaagggtg
     1021 cctttacctt ccaaggcagc atgattaaca tgccgctgct gaagcaggcc cagaacaccg
     1081 ttaccctggc caccagcatt aaggagaaac tcgagcacca ccaccaccac cactgagatc
     1141 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     1201 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     1261 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     1321 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     1381 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1441 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1501 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1561 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     1621 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     1681 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     1741 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     1801 tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa
     1861 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     1921 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     1981 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     2041 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     2101 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     2161 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
     2221 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     2281 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     2341 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     2401 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     2461 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     2521 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     2581 atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg
     2641 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca
     2701 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     2761 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     2821 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     2881 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     2941 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     3001 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     3061 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     3121 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     3181 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     3241 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     3301 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     3361 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     3421 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     3481 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     3541 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     3601 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     3661 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     3721 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     3781 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     3841 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     3901 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     3961 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     4021 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     4081 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     4141 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     4201 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     4261 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     4321 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     4381 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     4441 ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     4501 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     4561 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     4621 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     4681 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     4741 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     4801 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4861 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     4921 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     4981 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     5041 atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact cggtaatggc
     5101 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     5161 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     5221 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     5281 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     5341 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     5401 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     5461 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     5521 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     5581 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     5641 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     5701 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     5761 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     5821 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     5881 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     5941 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     6001 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     6061 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     6121 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     6181 cacctgtggc g
//
