LOCUS       sequence-274011-        5181 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SMT201 with mutated RBS for DHFR, in vivo assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5181)
  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 5181)
  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: SMT205
FEATURES             Location/Qualifiers
     source          1..5181
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(34..52,53..58,59..75,76..81,82..107)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    34..52
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    59..77
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             117..128
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             956..1000
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1038..1056)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1052..1099
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1320..1979)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1894..1913
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1980..2082)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2474..2491)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2608..3153)
                     /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(2688..2707)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3378..4460)
                     /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     4422..4441
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4461..4538)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4584..4603
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4584..4602
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4603..4627
                     /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             4671..5150
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
ORIGIN
        1 cttaagtcga acagaaagta atcgtattgt acatccctat cagtgataga gattgacatc
       61 cctatcagtg atagagatac tgagcacatc agcaggacgc actgaccgaa ttcattaaag
      121 aggagaaagg taccatatgg cagatctcat gaaacagtac ctggagctga tgcaaaaagt
      181 tctggatgag ggtacccaga aaaacgaccg cacgggtacc ggaacgctga gcatttttgg
      241 ccatcagatg cgttttaacc tgcaggacgg attcccgctg gttacgacca aacgctgcca
      301 cctgcgtagc attattcatg agctgctgtg gtttctgcaa ggtgacacta acatcgcgta
      361 tctgcacgaa aacaatgtga cgatctggga tgaatgggcc gatgaaaacg gcgatctggg
      421 cccagtgtat ggtaaacagt ggcgcgcctg gccaacgcca gatggccgcc acatcgatca
      481 gatcacgacc gtgctgaatc aactgaaaaa cgacccggac agccgccgca ttattgtttc
      541 cgcgtggaat gtgggtgaac tggataaaat ggcgctggcg ccgtgccatg cattcttcca
      601 gttttacgtt gcggacggta agctgagctg tcaactttat cagcgcagct gcgatgtttt
      661 cctcggcctg ccgttcaata tcgccagcta cgcgttactg gtgcatatga tggcgcagca
      721 gtgcgacctg gaggtgggtg attttgtctg gaccggcggt gatacccacc tgtacagcaa
      781 ccatatggac caaacgcatc tgcaattgag ccgtgagccg cgtccgctgc cgaaactgat
      841 tattaagcgc aaacccgaga gcatctttga ctatcgtttc gaagacttcg agattgaagg
      901 ctacgatccg cacccgggta tcaaagcgcc ggtggctatc taactcgagt ctggtaaaga
      961 aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg cagcttaatt
     1021 aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     1081 ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg tcacactgct
     1141 tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt aacgaccctg
     1201 ccctgaaccg acgaccgggt cgaatttgct ttcgaatttc tgccattcat ccgcttatta
     1261 tcacttattc aggcgtagca accaggcgtt taagggcacc aataactgcc ttaaaaaaat
     1321 tacgccccgc cctgccactc atcgcagtac tgttgtaatt cattaagcat tctgccgaca
     1381 tggaagccat cacaaacggc atgatgaacc tgaatcgcca gcggcatcag caccttgtcg
     1441 ccttgcgtat aatatttgcc catagtgaaa acgggggcga agaagttgtc catattggcc
     1501 acgtttaaat caaaactggt gaaactcacc cagggattgg ctgatacgaa aaacatattc
     1561 tcaataaacc ctttagggaa ataggccagg ttttcaccgt aacacgccac atcttgcgaa
     1621 tatatgtgta gaaactgccg gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt
     1681 tcagtttgct catggaaaac ggtgtaacaa gggtgaacac tatcccatat caccagctca
     1741 ccgtctttca ttgccatacg gaactccgga tgagcattca tcaggcgggc aagaatgtga
     1801 ataaaggccg gataaaactt gtgcttattt ttctttacgg tctttaaaaa ggccgtaata
     1861 tccagctgaa cggtctggtt ataggtacat tgagcaactg actgaaatgc ctcaaaatgt
     1921 tctttacgat gccattggga tatatcaacg gtggtatatc cagtgatttt tttctccatt
     1981 ttagcttcct tagctcctga aaatctcgat aactcaaaaa atacgcccgg tagtgatctt
     2041 atttcattat ggtgaaagtt ggaacctctt acgtgccgat caacgtctca ttttcgccaa
     2101 aagttggccc agggcttccc ggtatcaaca gggacaccag gatttattta ttctgcgaag
     2161 tgatcttccg tcacaggtat ttattcggcg caaagtgcgt cgggtgatgc tgccaactta
     2221 ctgatttagt gtatgatggt gtttttgagg tgctccagtg gcttctgttt ctatcagctg
     2281 tccctcctgt tcagctactg acggggtggt gcgtaacggc aaaagcaccg ccggacatca
     2341 gcgctagcgg agtgtatact ggcttactat gttggcactg atgagggtgt cagtgaagtg
     2401 cttcatgtgg caggagaaaa aaggctgcac cggtgcgtca gcagaatatg tgatacagga
     2461 tatattccgc ttcctcgctc actgactcgc tacgctcggt cgttcgactg cggcgagcgg
     2521 aaatggctta cgaacggggc ggagatttcc tggaagatgc caggaagata cttaacaggg
     2581 aagtgagagg gccgcggcaa agccgttttt ccataggctc cgcccccctg acaagcatca
     2641 cgaaatctga cgctcaaatc agtggtggcg aaacccgaca ggactataaa gataccaggc
     2701 gtttccccct tgcggctccc tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt
     2761 cattccgctg ttatggccgc gtttgtctca ttccacgcct gacactcagt tccgggtagg
     2821 cagttcgctc caagctggac tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct
     2881 tatccggtaa ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca ccactggcag
     2941 cagccactgg taattgattt agaggagtta gtcttgaagt catgcgccgg ttaaggctaa
     3001 actgaaagga caagttttgg tgactgcgct cctccaagcc agttacctcg gttcaaagag
     3061 ttggtagctc agagaacctt cgaaaaaccg ccctgcaagg cggttttttc gttttcagag
     3121 caagagatta cgcgcagacc aaaacgatct caagaagatc atcttattaa tcagataaaa
     3181 tatttctaga tttcagtgca atttatctct tcaaatgtag cacctgaagt cagccccata
     3241 cgatataagt tgtaattctc atgttagtca tgccccgcgc ccaccggaag gagctgactg
     3301 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     3361 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     3421 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     3481 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     3541 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     3601 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg
     3661 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     3721 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     3781 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     3841 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     3901 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     3961 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     4021 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     4081 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     4141 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     4201 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     4261 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     4321 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     4381 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     4441 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     4501 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     4561 atgcgactcc tgcattagga aattaatacg actcactata ggggaattgt gagcggataa
     4621 caattcccct gtagaaataa ttttgtttaa ctttaataat gagatatacc atgatcagtc
     4681 tgattgcggc gttagcggta gatcgcgtta tcggcatgga aaacgccatg ccgtggaacc
     4741 tgcctgccga tctcgcctgg tttaaacgca acaccttaaa taaacccgtg attatgggcc
     4801 gccatacctg ggaatcaatc ggtcgtccgt tgccaggacg caaaaatatt atcctcagca
     4861 gtcaaccggg tacggacgat cgcgtaacgt gggtgaagtc ggtggatgaa gccatcgcgg
     4921 cgtgtggtga cgtaccagaa atcatggtga ttggcggcgg tcgcgtttat gaacagttct
     4981 tgccaaaagc gcaaaaactg tatctgacgc atatcgacgc agaagtggaa ggcgacaccc
     5041 atttcccgga ttacgagccg gatgactggg aatcggtatt cagcgaattc cacgatgctg
     5101 atgcgcagaa ctctcacagc tattgctttg agattctgga gcggcggtaa caggcgtcga
     5161 caagcttgcg gccgcataat g
//
