LOCUS       sequence-229477-        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-FLAG
FEATURES             Location/Qualifiers
     source          1..8410
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    88..109
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(124..129,130..147,148..154)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    162..178
                     /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     167..189
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     186..202
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     186..202
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     221..241
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        223..241
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        269..495
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             496..676
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    723..848
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1341..1574
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1759..1803
                     /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     2101..2121
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        2410..2613
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2564..2584
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3425..3434
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3698..3721
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    3772..3889
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3938..4448
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4367..4385
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4470..5069
                     /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(4470..4489)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4966..4986
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5197..5430
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5502..5623
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5539..5558)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5593..5612
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5663..5798
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5818..5837)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5819..5837)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5847..5864)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5847..5863)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5856..5878)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6005..6460
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6092..6111)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6302..6323
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6486..6590
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6591..6659,6660..7451)
                     /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(6809..6828)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7622..8210
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8111..8130
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8364..8381
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
       61 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
      121 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
      181 tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag
      241 ggaacaaaag ctggagctgc aagcttaatg tagtcttatg caatactctt gtagtcttgc
      301 aacatggtaa cgatgagtta gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc
      361 cgattggtgg aagtaaggtg gtacgatcgt gccttattag gaaggcaaca gacgggtctg
      421 acatggattg gacgaaccac tgaattgccg cattgcagag atattgtatt taagtgccta
      481 gctcgataca taaacgggtc tctctggtta gaccagatct gagcctggga gctctctggc
      541 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
      601 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
      661 tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg
      721 agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc
      781 gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg
      841 agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc
      901 cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac
      961 gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa atactgggac
     1021 agctacaacc atcccttcag acaggatcag aagaacttag atcattatat aatacagtag
     1081 caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa gctttagaca
     1141 agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc
     1201 agacctggag gaggagatat gagggacaat tggagaagtg aattatataa atataaagta
     1261 gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga
     1321 gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc
     1381 actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata
     1441 gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc
     1501 acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag
     1561 gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg
     1621 ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg
     1681 atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa
     1741 tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt
     1801 ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata
     1861 gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt
     1921 aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac
     1981 aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta
     2041 gtgaacggat ctcgacggta tcgatcacga gactagcctc gagcggccgc ccccttcacc
     2101 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
     2161 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
     2221 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
     2281 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     2341 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     2401 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
     2461 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
     2521 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
     2581 cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca gatccgctag
     2641 catggcgcta cgtctgctgc ggagggcggc gcgcggagct gcggcggcgg cgctgctgag
     2701 gctgaaagcg tctctagcag ctgatatccc cagacttgga tatagttcct catcccatca
     2761 caagtacatc ccccggaggg cagtgctata cgttccagga aatgatgaaa agaaaataaa
     2821 gaagattcca tccctgaatg tagattgtgc agtgctcgac tgtgaggatg gagtggctgc
     2881 aaacaaaaag aatgaagctc gactgagaat tgtaaaaact cttgaagaca ttgatctggg
     2941 ccctactgaa aaatgtgtga gagtcaactc agtttccagt ggtctggcgg aagaagacct
     3001 agagaccctt ttgcaatccc gggtccttcc ttccagcctg atgctaccaa aggtggaaag
     3061 tcctgaagaa atccagtggt ttgcagacaa attttcattc cacttaaaag gccgaaaact
     3121 tgaacaacca atgaatttaa tcccttttgt ggaaactgca atgggtttgc tcaattttaa
     3181 ggcagtgtgt gaagaaaccc tgaaggtcgg gcctcaagta ggtctctttc tagatgcagt
     3241 cgtttttgga ggagaagact ttcgagccag cataggggcc acgtcgagta aagaaaccct
     3301 ggatattctc tacgcccggc aaaagattgt tgtcatagcg aaagcctttg gtctccaagc
     3361 catagatctg gtgtacattg actttcgaga tggagctggg ctgcttagac agtcacgaga
     3421 aggagccgcc atgggcttca ctggtaagca ggtgattcac cctaaccaaa ttgccgtggt
     3481 ccaggagcag ttttctcctt cccctgaaaa aattaagtgg gctgaagaac tgattgctgc
     3541 ctttaaagaa catcaacaat taggaaaggg ggcctttact ttccaaggga gtatgatcga
     3601 catgccatta ctgaagcagg cccagaacac tgttacgctt gccacctcca tcaaggaaaa
     3661 aaccgggggt ggatctggtg gatctggtgg atctatggat tacaaggatg acgatgacaa
     3721 gtaaggtacc gaattctcga cctcgagaca aatggcagta ttcatccaca attttaaaag
     3781 aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa tagcaacaga
     3841 catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaattttc gggtttatta
     3901 cagggacagc agagatccac tttggccgcg gctcgagggg gttggggttg cgccttttcc
     3961 aaggcagccc tgggtttgcg cagggacgcg gctgctctgg gcgtggttcc gggaaacgca
     4021 gcggcgccga ccctgggtct cgcacattct tcacgtccgt tcgcagcgtc acccggatct
     4081 tcgccgctac ccttgtgggc cccccggcga cgcttcctgc tccgccccta agtcgggaag
     4141 gttccttgcg gttcgcggcg tgccggacgt gacaaacgga agccgcacgt ctcactagta
     4201 ccctcgcaga cggacagcgc cagggagcaa tggcagcgcg ccgaccgcga tgggctgtgg
     4261 ccaatagcgg ctgctcagca gggcgcgccg agagcagcgg ccgggaaggg gcggtgcggg
     4321 aggcggggtg tggggcggta gtgtgggccc tgttcctgcc cgcgcggtgt tccgcattct
     4381 gcaagcctcc ggagcgcacg tcggcagtcg gctccctcgt tgaccgaatc accgacctct
     4441 ctccccaggg ggatccaccg gagcttacca tgaccgagta caagcccacg gtgcgcctcg
     4501 ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc
     4561 ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag
     4621 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
     4681 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
     4741 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
     4801 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct
     4861 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
     4921 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
     4981 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
     5041 ggtgcatgac ccgcaagccc ggtgcctgac gcccgcccca cgacccgcag cgcccgaccg
     5101 aaaggagcgc acgaccccat gcatcggtac ctttaagacc aatgacttac aaggcagctg
     5161 tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac
     5221 gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct
     5281 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
     5341 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
     5401 ccttttagtc agtgtggaaa atctctagca gtagtagttc atgtcatctt attattcagt
     5461 atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt
     5521 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     5581 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggctctagc
     5641 tatcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     5701 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     5761 gctattccag aagtagtgag gaggcttttt tggaggccta gggacgtacc caattcgccc
     5821 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
     5881 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     5941 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     6001 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     6061 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     6121 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     6181 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     6241 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     6301 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     6361 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     6421 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     6481 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     6541 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     6601 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     6661 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     6721 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     6781 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     6841 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     6901 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     6961 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     7021 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     7081 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     7141 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     7201 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     7261 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     7321 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     7381 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     7441 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     7501 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     7561 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     7621 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     7681 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     7741 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     7801 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     7861 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     7921 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     7981 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     8041 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     8101 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     8161 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     8221 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     8281 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     8341 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     8401 tacgcaaacc
//
