LOCUS       sequence-218596-        6591 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 6591)
  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 6591)
  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: His-PaIct
FEATURES             Location/Qualifiers
     source          1..6591
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     29..48
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        29..47
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    48..72
                     /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             87..109
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             120..137
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             147..164
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             171..215
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             1480..1497
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1550..1568)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1564..1611
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1648..2103
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1735..1754)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1945..1966
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2195..3010)
                     /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(2195..3010)
                     /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     2918..2937
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3002..3021
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3132..3720
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3621..3640
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3874..3891
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3906..4045
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3971..3990)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4131..4153
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4147..4338)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5147..6229)
                     /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     6191..6210
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6230..6307)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6513..6532
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 atcgagatcg atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
       61 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgc
      121 accatcatca tcatcattct tctggtctgg tgccacgcgg ttctggtatg aaagaaaccg
      181 ctgctgctaa attcgaacgc cagcacatgg acagcccaga tctgggtacc atgacccaga
      241 gtcatcgtcc gcgcccgctg gatggcatta ccgtggttag cctggagcat gccattgccg
      301 ccccgttttg cacacgtcag ctggccgatc tgggtgcccg tgtgatcaaa attgaacgtc
      361 cgggtgtggg cgattttgcc cgcggctatg atgaacgcgt taacggtctg agcagccatt
      421 ttgtgtggac aaaccgcagc aagcagagcc tgagcctgga tttaaagcag gccgaagcac
      481 tgccgctgct ggaaaaactg ctggccggcg ccgatgttct ggttcagaat ctggcaccgg
      541 gtgccgcagc ccgtctgggt ctgagctatg aagccttacg tgaacgcttt ccgcgtctga
      601 tcgtgtgcga tattagcggc tacggcgagg gtggtccgta cgagaagaag aaagcctacg
      661 acctgctgat tcagagcgaa ggcggttttc tgagcgtgac cggtggcgca ggcccgaatg
      721 aaatggccaa agccggctgc agcatcgcag acattgccgc cggcatgtac gcctacagta
      781 gtattctgag cgccctgctg ttacgcgaac gtaccggtga aggtagccgt atcgatgtga
      841 gcatgctgga aagtctggtg gaatggatgg gctacccgat gtattatgcc taccaggatg
      901 ccccgccgcc gccgcgcgcc ggtgccagcc atagtaccat ctacccttat ggcccgttcc
      961 cggcaggtga tggtggtaca gttatgctgg gtctgcagaa cgagcgtgag tggcgtgcat
     1021 tttgcgacaa agtgctgcgc cgtccggaac tggccgatga cgaacgcttc agcgccaatt
     1081 tcaaacgcag tgccaaccgt gaggaactgc gcgcactgat cgttgaggcc tttgcagcac
     1141 tgccggccgc agaagttgtt gcccgcctgg aacaggcaca aatcgccagc gcccatgtga
     1201 atgatatggc cggtgtttgg gcccatccgc aactgcaggc ccgtcagcgc tggacaagca
     1261 ttgatagccc ggccggtcgt ctgcctgcct tattaccgcc ggcaagcagc agtgcctttg
     1321 ttccgcgcat ggatgcagtg ccggcactgg gtggcgatag tgatgccctg ctggccgaac
     1381 tgggttgcac agccgccgat attcagcgtc tgcgcgcaac aggcactgtt tgatgatgag
     1441 aattcgagct ccgtcgacaa gcttgcggcc gcactcgagc accaccacca ccaccactga
     1501 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     1561 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     1621 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     1681 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     1741 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     1801 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     1861 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     1921 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     1981 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     2041 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     2101 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2161 acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat
     2221 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     2281 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     2341 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     2401 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     2461 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     2521 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     2581 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     2641 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     2701 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     2761 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     2821 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     2881 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     2941 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     3001 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
     3061 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     3121 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     3181 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3241 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     3301 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3361 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     3421 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     3481 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     3541 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     3601 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     3661 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     3721 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     3781 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     3841 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     3901 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     3961 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     4021 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     4081 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     4141 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     4201 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     4261 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     4321 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     4381 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     4441 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     4501 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     4561 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     4621 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     4681 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     4741 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     4801 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     4861 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     4921 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     4981 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     5041 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     5101 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     5161 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5221 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     5281 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     5341 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     5401 cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa
     5461 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     5521 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     5581 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     5641 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     5701 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     5761 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     5821 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     5881 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     5941 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     6001 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     6061 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     6121 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     6181 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     6241 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     6301 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     6361 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     6421 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     6481 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     6541 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag g
//
