LOCUS       sequence-218594-        8410 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8410)
  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 8410)
  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(D320A)-FLAG
FEATURES             Location/Qualifiers
     source          1..8410
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    243..364
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(280..299)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     334..353
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      404..539
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(559..578)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(560..578)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(588..605)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(588..604)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(597..619)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      746..1201
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(833..852)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1043..1064
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1227..1331
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1332..1400,1401..2192)
                     /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     complement(1550..1569)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2363..2951
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2852..2871
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3105..3122
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3239..3260
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3275..3280,3281..3298,3299..3305)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3313..3329
                     /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)."
     primer_bind     3318..3340
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3337..3353
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3337..3353
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3372..3392
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3374..3392
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3420..3646
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             3647..3827
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3874..3999
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    4492..4725
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4910..4954
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     primer_bind     5252..5272
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        5561..5764
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5715..5735
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      6576..6585
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6849..6872
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    6923..7040
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        7089..7599
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     7518..7536
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             7621..8220
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(7621..7640)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     8117..8137
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             join(8348..8410,1..171)
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
ORIGIN
        1 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
       61 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
      121 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt
      181 catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga
      241 ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
      301 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
      361 cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc
      421 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
      481 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
      541 agggacgtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
      601 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
      661 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
      721 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
      781 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
      841 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
      901 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
      961 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     1021 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     1081 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     1141 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     1201 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1261 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     1321 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     1381 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     1441 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     1501 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     1561 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     1621 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     1681 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     1741 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     1801 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     1861 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     1921 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     1981 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     2041 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     2101 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     2161 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     2221 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     2281 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     2341 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     2401 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     2461 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     2521 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     2581 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     2641 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     2701 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2761 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2821 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     2881 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     2941 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3001 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     3061 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     3121 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     3181 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     3241 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     3301 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     3361 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat
     3421 gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc
     3481 cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg
     3541 tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc
     3601 gcattgcaga gatattgtat ttaagtgcct agctcgatac ataaacgggt ctctctggtt
     3661 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     3721 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     3781 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac
     3841 agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct
     3901 gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact
     3961 agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt
     4021 agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa
     4081 catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa
     4141 acatcagaag gctgtagaca aatactggga cagctacaac catcccttca gacaggatca
     4201 gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata
     4261 gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag
     4321 accaccgcac agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa
     4381 ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc
     4441 caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt
     4501 gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac
     4561 ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc
     4621 tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc
     4681 aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg
     4741 ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc
     4801 tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta
     4861 cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca
     4921 agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg
     4981 gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt
     5041 ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca
     5101 gacccacctc ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg
     5161 agagagagac agagacagat ccattcgatt agtgaacgga tctcgacggt atcgatcacg
     5221 agactagcct cgagcggccg cccccttcac cgagggccta tttcccatga ttccttcata
     5281 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
     5341 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     5401 tttaaaatta tgttttaaaa tggactatca tatgccaagt acgcccccta ttgacgtcaa
     5461 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
     5521 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
     5581 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
     5641 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
     5701 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
     5761 agctggttta gtgaaccgtc agatccgcta gcatggcgct acgtctgctg cggagggcgg
     5821 cgcgcggagc tgcggcggcg gcgctgctga ggctgaaagc gtctctagca gctgatatcc
     5881 ccagacttgg atatagttcc tcatcccatc acaagtacat cccccggagg gcagtgctat
     5941 acgttccagg aaatgatgaa aagaaaataa agaagattcc atccctgaat gtagattgtg
     6001 cagtgctcga ctgtgaggat ggagtggctg caaacaaaaa gaatgaagct cgactgagaa
     6061 ttgtaaaaac tcttgaagac attgatctgg gccctactga aaaatgtgtg agagtcaact
     6121 cagtttccag tggtctggcg gaagaagacc tagagaccct tttgcaatcc cgggtccttc
     6181 cttccagcct gatgctacca aaggtggaaa gtcctgaaga aatccagtgg tttgcagaca
     6241 aattttcatt ccacttaaaa ggccgaaaac ttgaacaacc aatgaattta atcccttttg
     6301 tggaaactgc aatgggtttg ctcaatttta aggcagtgtg tgaagaaacc ctgaaggtcg
     6361 ggcctcaagt aggtctcttt ctagatgcag tcgtttttgg aggagaagac tttcgagcca
     6421 gcataggggc cacgtcgagt aaagaaaccc tggatattct ctacgcccgg caaaagattg
     6481 ttgtcatagc gaaagccttt ggtctccaag ccatagatct ggtgtacatt gactttcgag
     6541 atggagctgg gctgcttaga cagtcacgag aaggagccgc catgggcttc actggtaagc
     6601 aggtgattca ccctaaccaa attgccgtgg tccaggagca gttttctcct tcccctgaaa
     6661 aaattaagtg ggctgaagaa ctgattgctg cctttaaaga acatcaacaa ttaggaaagg
     6721 gggcctttac tttccaaggg agtatgatcg ccatgccatt actgaagcag gcccagaaca
     6781 ctgttacgct tgccacctcc atcaaggaaa aaaccggggg tggatctggt ggatctggtg
     6841 gatctatgga ttacaaggat gacgatgaca agtaaggtac cgaattctcg acctcgagac
     6901 aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag
     6961 gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa
     7021 ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggccgc
     7081 ggctcgaggg ggttggggtt gcgccttttc caaggcagcc ctgggtttgc gcagggacgc
     7141 ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg accctgggtc tcgcacattc
     7201 ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg ccccccggcg
     7261 acgcttcctg ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg
     7321 tgacaaacgg aagccgcacg tctcactagt accctcgcag acggacagcg ccagggagca
     7381 atggcagcgc gccgaccgcg atgggctgtg gccaatagcg gctgctcagc agggcgcgcc
     7441 gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc
     7501 ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc
     7561 ggctccctcg ttgaccgaat caccgacctc tctccccagg gggatccacc ggagcttacc
     7621 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     7681 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     7741 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     7801 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     7861 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     7921 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     7981 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
     8041 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     8101 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     8161 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga
     8221 cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccca tgcatcggta
     8281 cctttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag
     8341 gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact
     8401 gggtctctct
//
