LOCUS       sequence-209280-        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"
     promoter        131..334
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     285..305
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1146..1155
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1419..1442
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    1493..1610
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1659..2169
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2088..2106
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2191..2790
                     /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(2191..2210)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2687..2707
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2918..3151
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3223..3344
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3260..3279)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3314..3333
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3384..3519
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3539..3558)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3540..3558)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3568..3585)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3568..3584)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3577..3599)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3726..4181
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3813..3832)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4023..4044
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4207..4311
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4312..4380,4381..5172)
                     /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(4530..4549)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5343..5931
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5832..5851
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6085..6102
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6219..6240
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6255..6260,6261..6278,6279..6285)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6293..6309
                     /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     6298..6320
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6317..6333
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6317..6333
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6352..6372
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6354..6372
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6400..6626
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6627..6807
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6854..6979
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7472..7705
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7890..7934
                     /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     8232..8252
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
       61 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      121 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      181 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      241 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      301 gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta
      361 gcatggcgct acgtctgctg cggagggcgg cgcgcggagc tgcggcggcg gcgctgctga
      421 ggctgaaagc gtctctagca gctgatatcc ccagacttgg atatagttcc tcatcccatc
      481 acaagtacat cccccggagg gcagtgctat acgttccagg aaatgatgaa aagaaaataa
      541 agaagattcc atccctgaat gtagattgtg cagtgctcga ctgtgaggat ggagtggctg
      601 caaacaaaaa gaatgaagct cgactgagaa ttgtaaaaac tcttgaagac attgatctgg
      661 gccctactga aaaatgtgtg agagtcaact cagtttccag tggtctggcg gaagaagacc
      721 tagagaccct tttgcaatcc cgggtccttc cttccagcct gatgctacca aaggtggaaa
      781 gtcctgaaga aatccagtgg tttgcagaca aattttcatt ccacttaaaa ggccgaaaac
      841 ttgaacaacc aatgaattta atcccttttg tggaaactgc aatgggtttg ctcaatttta
      901 aggcagtgtg tgaagaaacc ctgaaggtcg ggcctcaagt aggtctcttt ctagatgcag
      961 tcgtttttgg aggagaagac tttcgagcca gcataggggc cacgtcgagt aaagaaaccc
     1021 tggatattct ctacgcccgg caaaagattg ttgtcatagc gaaagccttt ggtctccaag
     1081 ccatagatct ggtgtacatt gactttcgag atggagctgg gctgcttaga cagtcacgag
     1141 aaggagccgc catgggcttc actggtaagc aggtgattca ccctaaccaa attgccgtgg
     1201 tccaggagca gttttctcct tcccctgaaa aaattaagtg ggctgaagaa ctgattgctg
     1261 cctttaaaga acatcaacaa ttaggaaagg gggcctttac tttccaaggg agtatgatcg
     1321 ccatgccatt actgaagcag gcccagaaca ctgttacgct tgccacctcc atcaaggaaa
     1381 aaaccggggg tggatctggt ggatctggtg gatctatgga ttacaaggat gacgatgaca
     1441 agtaaggtac cgaattctcg acctcgagac aaatggcagt attcatccac aattttaaaa
     1501 gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag
     1561 acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt
     1621 acagggacag cagagatcca ctttggccgc ggctcgaggg ggttggggtt gcgccttttc
     1681 caaggcagcc ctgggtttgc gcagggacgc ggctgctctg ggcgtggttc cgggaaacgc
     1741 agcggcgccg accctgggtc tcgcacattc ttcacgtccg ttcgcagcgt cacccggatc
     1801 ttcgccgcta cccttgtggg ccccccggcg acgcttcctg ctccgcccct aagtcgggaa
     1861 ggttccttgc ggttcgcggc gtgccggacg tgacaaacgg aagccgcacg tctcactagt
     1921 accctcgcag acggacagcg ccagggagca atggcagcgc gccgaccgcg atgggctgtg
     1981 gccaatagcg gctgctcagc agggcgcgcc gagagcagcg gccgggaagg ggcggtgcgg
     2041 gaggcggggt gtggggcggt agtgtgggcc ctgttcctgc ccgcgcggtg ttccgcattc
     2101 tgcaagcctc cggagcgcac gtcggcagtc ggctccctcg ttgaccgaat caccgacctc
     2161 tctccccagg gggatccacc ggagcttacc atgaccgagt acaagcccac ggtgcgcctc
     2221 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     2281 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     2341 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     2401 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     2461 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     2521 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     2581 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     2641 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     2701 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     2761 tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc acgacccgca gcgcccgacc
     2821 gaaaggagcg cacgacccca tgcatcggta cctttaagac caatgactta caaggcagct
     2881 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     2941 cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     3001 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     3061 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     3121 cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag
     3181 tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct
     3241 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     3301 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag
     3361 ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     3421 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     3481 agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc
     3541 ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga
     3601 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     3661 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     3721 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     3781 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3841 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3901 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     3961 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     4021 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     4081 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     4141 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     4201 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4261 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4321 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4381 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     4441 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     4501 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     4561 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     4621 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     4681 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     4741 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     4801 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     4861 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     4921 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     4981 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     5041 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5101 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5161 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     5221 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     5281 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5341 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     5401 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     5461 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     5521 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     5581 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     5641 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     5701 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     5761 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     5821 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     5881 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     5941 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     6001 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     6061 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     6121 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     6181 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     6241 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     6301 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac
     6361 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct
     6421 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc
     6481 accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac
     6541 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat
     6601 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg
     6661 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     6721 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     6781 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg
     6841 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg
     6901 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag
     6961 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat
     7021 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag
     7081 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca
     7141 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata
     7201 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga
     7261 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc
     7321 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata
     7381 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag
     7441 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag
     7501 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat
     7561 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc
     7621 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa
     7681 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca
     7741 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc
     7801 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct
     7861 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata
     7921 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat
     7981 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag
     8041 tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga
     8101 ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat
     8161 ccattcgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg
     8221 cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     8281 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
     8341 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     8401 tggactatca
//
