LOCUS       sequence-313942-        5182 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual expression of DHFR and TYMS, in vivo assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5182)
  AUTHORS   Thompson S, Zhang Y, Ingle C, Reynolds KA, Kortemme T
  TITLE     Altered expression of a quality control protease in E. coli reshapes
            the in vivo mutational landscape of a model enzyme.
  JOURNAL   eLife. 2020 Jul 23;9. pii: 53476. doi: 10.7554/eLife.53476.
  PUBMED    32701056
REFERENCE   2  (bases 1 to 5182)
  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"; journalName: "eLife."; 
            date: "2020-07-23"; volume: "9. pii: 53476. doi"; pages: " 
            10.7554/eLife.53476"
COMMENT     Sequence Label: SMT101
FEATURES             Location/Qualifiers
     source          1..5182
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     CDS             906..950
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(988..1006)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1002..1049
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1270..1929)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1844..1863
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1930..2032)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2424..2441)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2558..3103)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(2638..2657)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3328..4410)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4372..4391
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4411..4488)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        4534..4552
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4553..4577
                     /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)."
     CDS             4621..5100
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttccccatct tagtatatta
       61 gttaagtata agaaggagat atacatatgg cagatctcat gaaacagtac ctggagctga
      121 tgcaaaaagt tctggatgag ggtacccaga aaaacgaccg cacgggtacc ggaacgctga
      181 gcatttttgg ccatcagatg cgttttaacc tgcaggacgg attcccgctg gttacgacca
      241 aacgctgcca cctgcgtagc attattcatg agctgctgtg gtttctgcaa ggtgacacta
      301 acatcgcgta tctgcacgaa aacaatgtga cgatctggga tgaatgggcc gatgaaaacg
      361 gcgatctggg cccagtgtat ggtaaacagt ggcgcgcctg gccaacgcca gatggccgcc
      421 acatcgatca gatcacgacc gtgctgaatc aactgaaaaa cgacccggac agccgccgca
      481 ttattgtttc cgcgtggaat gtgggtgaac tggataaaat ggcgctggcg ccgtgccatg
      541 cattcttcca gttttacgtt gcggacggta agctgagctg tcaactttat cagcgcagct
      601 gcgatgtttt cctcggcctg ccgttcaata tcgccagcta cgcgttactg gtgcatatga
      661 tggcgcagca gtgcgacctg gaggtgggtg attttgtctg gaccggcggt gatacccacc
      721 tgtacagcaa ccatatggac caaacgcatc tgcaattgag ccgtgagccg cgtccgctgc
      781 cgaaactgat tattaagcgc aaacccgaga gcatctttga ctatcgtttc gaagacttcg
      841 agattgaagg ctacgatccg cacccgggta tcaaagcgcc ggtggctatc taactcgagt
      901 ctggtaaaga aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg
      961 cagcttaatt aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     1021 cctctaaacg ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg
     1081 tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt
     1141 aacgaccctg ccctgaaccg acgaccgggt cgaatttgct ttcgaatttc tgccattcat
     1201 ccgcttatta tcacttattc aggcgtagca accaggcgtt taagggcacc aataactgcc
     1261 ttaaaaaaat tacgccccgc cctgccactc atcgcagtac tgttgtaatt cattaagcat
     1321 tctgccgaca tggaagccat cacaaacggc atgatgaacc tgaatcgcca gcggcatcag
     1381 caccttgtcg ccttgcgtat aatatttgcc catagtgaaa acgggggcga agaagttgtc
     1441 catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg ctgagacgaa
     1501 aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt aacacgccac
     1561 atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac tccagagcga
     1621 tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac tatcccatat
     1681 caccagctca ccgtctttca ttgccatacg gaactccgga tgagcattca tcaggcgggc
     1741 aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg tctttaaaaa
     1801 ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg actgaaatgc
     1861 ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc cagtgatttt
     1921 tttctccatt ttagcttcct tagctcctga aaatctcgat aactcaaaaa atacgcccgg
     1981 tagtgatctt atttcattat ggtgaaagtt ggaacctctt acgtgccgat caacgtctca
     2041 ttttcgccaa aagttggccc agggcttccc ggtatcaaca gggacaccag gatttattta
     2101 ttctgcgaag tgatcttccg tcacaggtat ttattcggcg caaagtgcgt cgggtgatgc
     2161 tgccaactta ctgatttagt gtatgatggt gtttttgagg tgctccagtg gcttctgttt
     2221 ctatcagctg tccctcctgt tcagctactg acggggtggt gcgtaacggc aaaagcaccg
     2281 ccggacatca gcgctagcgg agtgtatact ggcttactat gttggcactg atgagggtgt
     2341 cagtgaagtg cttcatgtgg caggagaaaa aaggctgcac cggtgcgtca gcagaatatg
     2401 tgatacagga tatattccgc ttcctcgctc actgactcgc tacgctcggt cgttcgactg
     2461 cggcgagcgg aaatggctta cgaacggggc ggagatttcc tggaagatgc caggaagata
     2521 cttaacaggg aagtgagagg gccgcggcaa agccgttttt ccataggctc cgcccccctg
     2581 acaagcatca cgaaatctga cgctcaaatc agtggtggcg aaacccgaca ggactataaa
     2641 gataccaggc gtttccccct ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt
     2701 ttaccggtgt cattccgctg ttatggccgc gtttgtctca ttccacgcct gacactcagt
     2761 tccgggtagg cagttcgctc caagctggac tgtatgcacg aaccccccgt tcagtccgac
     2821 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca
     2881 ccactggcag cagccactgg taattgattt agaggagtta gtcttgaagt catgcgccgg
     2941 ttaaggctaa actgaaagga caagttttgg tgactgcgct cctccaagcc agttacctcg
     3001 gttcaaagag ttggtagctc agagaacctt cgaaaaaccg ccctgcaagg cggttttttc
     3061 gttttcagag caagagatta cgcgcagacc aaaacgatct caagaagatc atcttattaa
     3121 tcagataaaa tatttctaga tttcagtgca atttatctct tcaaatgtag cacctgaagt
     3181 cagccccata cgatataagt tgtaattctc atgttagtca tgccccgcgc ccaccggaag
     3241 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     3301 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     3361 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3421 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     3481 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     3541 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     3601 taccgagatg tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     3661 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     3721 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     3781 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     3841 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     3901 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     3961 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     4021 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     4081 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     4141 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     4201 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     4261 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     4321 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     4381 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     4441 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     4501 gctctccctt atgcgactcc tgcattagga aattaatacg actcactata ggggaattgt
     4561 gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag gagatatacc
     4621 atgatcagtc tgattgcggc gttagcggta gatcgcgtta tcggcatgga aaacgccatg
     4681 ccgtggaacc tgcctgccga tctcgcctgg tttaaacgca acaccttaaa taaacccgtg
     4741 attatgggcc gccatacctg ggaatcaatc ggtcgtccgt tgccaggacg caaaaatatt
     4801 atcctcagca gtcaaccggg tacggacgat cgcgtaacgt gggtgaagtc ggtggatgaa
     4861 gccatcgcgg cgtgtggtga cgtaccagaa atcatggtga ttggcggcgg tcgcgtttat
     4921 gaacagttct tgccaaaagc gcaaaaactg tatctgacgc atatcgacgc agaagtggaa
     4981 ggcgacaccc atttcccgga ttacgagccg gatgactggg aatcggtatt cagcgaattc
     5041 cacgatgctg atgcgcagaa ctctcacagc tattgctttg agattctgga gcggcggtaa
     5101 caggcgtcga caagcttgcg gccgcataat gcttaagtcg aacagaaagt aatcgtattg
     5161 tacacggccg cataatcgaa at
//
