LOCUS       sequence-373745-        1671 bp    DNA     linear   UNA 14-APR-2025
DEFINITION  Expresses an N-terminal enhanced destabilizing domain (DD) version 
            of E. coli DHFR with higher basal turnover in mammalian systems. 
            Contains missense mutations W74R/T113S/E120D/Q146L..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 1671)
  AUTHORS   Nakahara E, Mullapudi V, Collier GE, Joachimiak LA, Hulleman JD
  TITLE     Development of a New DHFR-Based Destabilizing Domain with Enhanced 
            Basal Turnover and Applicability in Mammalian Systems.
  JOURNAL   ACS Chem Biol. 2022 Sep 19. doi: 10.1021/acschembio.2c00518.
  PUBMED    36122928
REFERENCE   2  (bases 1 to 1671)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Chem 
            Biol. 2022 Sep 19. doi: 10.1021/acschembio.2c00518."
COMMENT     Sequence Label: pcDNA C12 ecDHFR DD YFP HA
FEATURES             Location/Qualifiers
     source          1..1671
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     249..268
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        249..267
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    297..321
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             365..841
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPGTDDRVTRVKSVDEAIAACGDVPEIMVIGGGRVYEQFL
                     PKAQKLYLSHIDAEVDGDTHFPDYEPDDWESVFSEFHDADALNSHSYCFEILERR"
     CDS             join(848..850,851..1561)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(890..911)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1151..1170)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1498..1519
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1568..1594
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1600..1641
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1651..1668
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
      241 atcgaaatta atacgactca ctatagggag acccaagctg gctagttaag ctatcaacaa
      301 gtttgtacaa aaaagcaggc tttaaaggaa ccaattcagt cgactggatc cggtaccggc
      361 caccatgatc agtctgattg cggcgttagc ggtagatcgc gttatcggca tggaaaacgc
      421 catgccgtgg aacctgcctg ccgatctcgc ctggtttaaa cgcaacacct taaataaacc
      481 cgtgattatg ggccgccata cctgggaatc aatcggtcgt ccgttgccag gacgcaaaaa
      541 tattatcctc agcagtcaac cgggtacgga cgatcgcgta acgcgggtga agtcggtgga
      601 tgaagccatc gcggcgtgtg gtgacgtacc agaaatcatg gtgattggcg gcggtcgcgt
      661 ttatgaacag ttcttgccaa aagcgcaaaa actgtatctg tcgcatatcg acgcagaagt
      721 ggatggcgac acccatttcc cggattacga gccggatgac tgggaatcgg tattcagcga
      781 attccacgat gccgatgcgc tgaactctca cagctattgc tttgagattc tggagcggcg
      841 ggcatgcgtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
      901 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
      961 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     1021 caccctcgtg accaccttcg gctacggcct gcagtgcttc gcccgctacc ccgaccacat
     1081 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     1141 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1201 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1261 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1321 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1381 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1441 ccactacctg agctaccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     1501 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1561 ggtcgactat ccgtacgacg taccagacta cgcataaaag gtaagcctat ccctaaccct
     1621 ctcctcggtc tcgattctac gcgtaccggt catcatcacc atcaccattg a
//
