LOCUS       sequence-458981-        6036 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient overexpression of mouse FEM1B in mammalian cells. Mutation
            abolishes substrate binding..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6036)
  AUTHORS   Manford AG, Rodriguez-Perez F, Shih KY, Shi Z, Berdan CA, Choe M, 
            Titov DV, Nomura DK, Rape M
  TITLE     A Cellular Mechanism to Detect and Alleviate Reductive Stress.
  JOURNAL   Cell. 2020 Oct 1;183(1):46-61.e21. doi: 10.1016/j.cell.2020.08.034. 
            Epub 2020 Sep 16.
  PUBMED    32941802
REFERENCE   2  (bases 1 to 6036)
  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.2020.08.034"; journalName: "Cell"; date: "2020-10-1-
            1"; volume: "183"; issue: "1"; pages: "46-61.e21"
COMMENT     Sequence Label: pCS2+ 3xFLAG-FEM1B C186S
FEATURES             Location/Qualifiers
     source          1..6036
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     350..369
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(588..692)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(718..1173)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(855..876)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1067..1086
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1244..2228
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        2263..2281
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2263..2280
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      2313..2322
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2322..2387
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    4316..4450
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4340..4359)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4394..4413
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4563..4583)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4563..4581)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4602..4618)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4602..4618)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4615..4637)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4626..4642
                     /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)."
     promoter        complement(join(4650..4656,4657..4674,4675..4680))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4695..4716
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4833..4850)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5004..5592)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5084..5103)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5763..6036,1..518,519..587))
                     /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"
ORIGIN
        1 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
       61 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
      121 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
      181 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
      241 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
      301 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
      361 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
      421 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
      481 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
      541 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
      601 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
      661 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
      721 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
      781 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
      841 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
      901 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
      961 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     1021 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     1081 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     1141 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact
     1201 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagtcgacc gccaattcaa
     1261 tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt gccaattcaa
     1321 tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca gccaattcaa
     1381 tatggcggac ttggcaccat gccaattcaa tatggcggac ctggcactgt gccaactggg
     1441 gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg tgccaagtcc
     1501 gccatattga attggcatgg tgccaataat ggcggccata ttggctatat gccaggatca
     1561 atatataggc aatatccaat atggccctat gccaatatgg ctattggcca ggttcaatac
     1621 tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt gacgtagata
     1681 gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac cgcccatagc
     1741 cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac gtcatatggg
     1801 cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta aatggcccgc
     1861 ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa tgggatggct
     1921 cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg taaatggccc
     1981 acttggcagt acatcaatat ctattaatag taacttggca agtacattac tattggaagt
     2041 acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg gcccgcgatg
     2101 gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa atgggcgttc
     2161 cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc aatgctcgtt
     2221 tagggaaccg ccattctgcc tggggacgtc ggagcaagct tgatttaggt gacactatag
     2281 aatacaagct acttgttctt tttgcaggat ccgccaccat ggactacaaa gaccatgacg
     2341 gtgattataa agatcatgac atcgattaca aggatgacga tgacaagggc ggaggagtcg
     2401 acatggaggg cctggctggc tatgtgtaca aggcggccag cgagggcaag gtgctcactc
     2461 tggctgcctt gctccttaac cggtcagaga gcgatatccg ctacctgctg ggctatgtca
     2521 gtcagcaggg aggacagcgc tccacacccc tcatcatcgc agcccgcaat gggcacgcca
     2581 aggtggtgcg cttgctgtta gaacactacc gtgtgcagac ccagcagacc ggcaccgtcc
     2641 gcttcgacgg gtatgtcatt gacggtgcca ctgctctttg gtgtgctgcg ggagccggac
     2701 attttgaagt tgttaaactt ctagtcagcc atggagccaa cgtgaaccac accacagtca
     2761 ctaactcaac tccattgcgg gcagcatgct ttgatggcag actggacatt gtgaaatatt
     2821 tggttgaaaa taatgccaat atcagcattg ccaacaagta tgacaacacc tgcctaatga
     2881 tcgcagcata taaagggcac actgatgtgg tcagatacct cttagaacaa cgtgctgatc
     2941 ccaatgctaa agcacactct ggagccacag ctttgcactt tgcagccgaa gctggtcaca
     3001 ttgacattgt gaaagaactg ataaaatgga gagctgcaat agtggtgaat ggccatggga
     3061 tgacaccatt gaaggtggcc gctgaaagct gtaaagctga tgtcgttgaa ctgttgctct
     3121 ctcatgctga ttgtgaccgc agaagtcgga ttgaagcctt ggagctcttg ggtgcctcct
     3181 ttgcaaatga tcgtgagaac tatgacatca tgaagacata ccactattta tatttagcta
     3241 tgttggagag atttcaggat ggtgacaaca ttcttgaaaa agaggttctc ccacccatcc
     3301 atgcttatgg gaacagaact gagtgtagga acccacagga attggaggct attcggcaag
     3361 acagagatgc tcttcacatg gagggcctta tagttcggga acggatttta ggtgctgaca
     3421 acattgatgt ttcccacccc atcatttaca gaggggctgt ctatgctgat aacatggagt
     3481 tcgagcagtg catcaaattg tggctccacg cactacacct gaggcagaaa ggtaacagga
     3541 atacccacaa agatctgctt cggtttgctc aagtcttctc tcagatgata cacctcaatg
     3601 aagctgtgaa ggccccagac atagagtgcg ttttgagatg cagtgtcttg gaaatagagc
     3661 agagcatgaa cagagttaaa aatatctccg atgccgacgt ccacagtgcc atggataact
     3721 atgagtgtaa cctctatacc tttctgtacc tcgtgtgcat ctccaccaag acacagtgta
     3781 gcgaagaaga tcagtgcaga attaacaagc agatctacaa cctgattcac ctggacccca
     3841 gaacacggga aggtttttcc ttgctacacc tggctgtcaa ctcgaacaca ccagttgatg
     3901 atttccatac taacgatgtc tgcagctttc ccaacgctct ggtcacaaag ctcctgctgg
     3961 actgtggcgc tgaggtaaat gcagtggaca atgaggggaa cagtgccctc cacattatcg
     4021 tccagtacaa caggcccatc agtgattttc tgactttgca ctccatcatc atcagccttg
     4081 tggaggctgg agctcacacc gacatgacaa acaagcagaa taaaactccg ctagacaaaa
     4141 gtacaactgg ggtgtctgaa atactactta aaactcagat gaagatgagc ctcaagtgcc
     4201 tggctgcccg agcagttcgg gctaatgaca ttaactacca agaccagatc ccccggactc
     4261 ttgaagagtt tgttggattt cattaatcta gaactatagt gagtcgtatt acgtagatcc
     4321 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     4381 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     4441 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     4501 ggaggttttt taattcgcgg ccgcggcgcc aatgcattgg gcccggtacc cagcttttgt
     4561 tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg
     4621 tgaaattgtt atccgctcac aattccacac aacatacgag ccgggagcat aaagtgtaaa
     4681 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4741 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4801 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4861 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4921 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4981 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5041 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5101 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5161 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     5221 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5281 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5341 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5401 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     5461 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5521 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5581 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5641 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5701 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5761 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5821 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5881 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5941 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6001 cagtctatta attgttgccg ggaagctaga gtaagt
//
