LOCUS       sequence-225197-        6431 bp    DNA     circular SYN 15-APR-2025
DEFINITION  bacterial expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6431)
  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 6431)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: CaMCLC-His
FEATURES             Location/Qualifiers
     source          1..6431
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..41
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        22..40
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    41..65
                     /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             80..102
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1184..1201
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1254..1272)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1268..1315
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1352..1807
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1439..1458)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1649..1670
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1833..1937
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1938..2006,2007..2798)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2156..2175)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2969..3557
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3458..3477
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3711..3728
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3743..3882
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3808..3827)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3968..3990
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3984..4175)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4984..6066)
                     /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     6028..6047
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6067..6144)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6350..6369
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
       61 attcccctct agaaataatt ttgtttaact ttaagaagga gatatacata tgcgtaaact
      121 ggcccataac ttttataaac cgctggcaat tggagcaccg gaaccgatcc gcgaactgcc
      181 ggttcgtccg gaacgtgtgg ttcatttctt tccgccgcac gtggaaaaaa ttcgtgctcg
      241 catcccggaa gttgctaaac aggttgatgt cctgtgcggc aacctggaag atgcaattcc
      301 gatggacgct aaagaagcgg cccgtaatgg ttttatcgaa gtcgtgaaag caaccgattt
      361 cggcgacacg gctctgtggg tgcgcgttaa cgcgctgaac agcccgtggg tgctggatga
      421 cattgccgaa atcgttgcag ctgtcggtaa caaactggat gtcattatga tcccgaaagt
      481 ggaaggcccg tgggatattc actttgttga ccagtacctg gcgctgctgg aagcccgtca
      541 tcaaatcaaa aaaccgattc tgatccacgc gctgctggaa accgcccagg gtatggtgaa
      601 tctggaagaa attgcgggtg ccagcccgcg tatgcacggt ttctctctgg gtccggcgga
      661 tctggcagcc tcgcgtggta tgaaaaccac gcgcgttggc ggtggccacc cgttttatgg
      721 tgtcctggcc gatccgcagg aaggccaagc agaacgtccg ttctatcagc aggatctgtg
      781 gcattacacc attgcgcgta tggttgacgt ggcagttgct cacggtctgc gtgcctttta
      841 cggtccgttc ggcgatatca aagacgaagc agcttgcgaa gcacagtttc gcaatgcttt
      901 cctgctgggt tgtacgggcg catggagtct ggctccgaac caaattccga tcgcaaaacg
      961 tgtcttttcc ccggatgtga atgaagttct gttcgcgaaa cgcattctgg aagccatgcc
     1021 ggatggttct ggcgtggcga tgatcgacgg taaaatgcag gatgacgcga cgtggaaaca
     1081 agccaaagtc attgtggatc tggcgcgtat gatcgccaag aaggacccgg acctggcgca
     1141 ggcctatggc ctgggcggtg gttcgggcgg tggctctctc gagcaccacc accaccacca
     1201 ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     1261 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     1321 aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     1381 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     1441 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     1501 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     1561 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     1621 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     1681 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     1741 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     1801 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1861 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     1921 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     1981 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     2041 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     2101 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     2161 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     2221 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     2281 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     2341 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     2401 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     2461 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     2521 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     2581 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     2641 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     2701 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     2761 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     2821 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     2881 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2941 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     3001 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     3061 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     3121 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     3181 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     3241 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3301 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3361 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3421 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3481 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3541 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3601 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3661 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     3721 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3781 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3841 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     3901 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     3961 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     4021 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     4081 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     4141 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     4201 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     4261 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     4321 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     4381 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     4441 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     4501 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     4561 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     4621 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg
     4681 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     4741 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     4801 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     4861 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     4921 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     4981 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     5041 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     5101 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     5161 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     5221 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     5281 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     5341 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     5401 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     5461 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     5521 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     5581 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     5641 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     5701 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     5761 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     5821 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     5881 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     5941 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     6001 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     6061 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     6121 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     6181 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     6241 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     6301 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     6361 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     6421 cgtagaggat c
//
