LOCUS       sequence-313944-        5205 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SMT205 with DHFR-FLAG-tag, western blot.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5205)
  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 5205)
  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: SMT215
FEATURES             Location/Qualifiers
     source          1..5205
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..19,20..25,26..42,43..48,49..74)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    1..19
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    26..44
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             84..95
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             923..967
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1005..1023)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1019..1066
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1287..1946)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1861..1880
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1947..2049)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2441..2458)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2575..3120)
                     /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(2655..2674)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3345..4427)
                     /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     4389..4408
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4428..4505)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4551..4570
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4551..4569
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4570..4594
                     /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             4638..5114
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             5133..5156
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
ORIGIN
        1 tccctatcag tgatagagat tgacatccct atcagtgata gagatactga gcacatcagc
       61 aggacgcact gaccgaattc attaaagagg agaaaggtac catatggcag atctcatgaa
      121 acagtacctg gagctgatgc aaaaagttct ggatgagggt acccagaaaa acgaccgcac
      181 gggtaccgga acgctgagca tttttggcca tcagatgcgt tttaacctgc aggacggatt
      241 cccgctggtt acgaccaaac gctgccacct gcgtagcatt attcatgagc tgctgtggtt
      301 tctgcaaggt gacactaaca tcgcgtatct gcacgaaaac aatgtgacga tctgggatga
      361 atgggccgat gaaaacggcg atctgggccc agtgtatggt aaacagtggc gcgcctggcc
      421 aacgccagat ggccgccaca tcgatcagat cacgaccgtg ctgaatcaac tgaaaaacga
      481 cccggacagc cgccgcatta ttgtttccgc gtggaatgtg ggtgaactgg ataaaatggc
      541 gctggcgccg tgccatgcat tcttccagtt ttacgttgcg gacggtaagc tgagctgtca
      601 actttatcag cgcagctgcg atgttttcct cggcctgccg ttcaatatcg ccagctacgc
      661 gttactggtg catatgatgg cgcagcagtg cgacctggag gtgggtgatt ttgtctggac
      721 cggcggtgat acccacctgt acagcaacca tatggaccaa acgcatctgc aattgagccg
      781 tgagccgcgt ccgctgccga aactgattat taagcgcaaa cccgagagca tctttgacta
      841 tcgtttcgaa gacttcgaga ttgaaggcta cgatccgcac ccgggtatca aagcgccggt
      901 ggctatctaa ctcgagtctg gtaaagaaac cgctgctgcg aaatttgaac gccagcacat
      961 ggactcgtct actagcgcag cttaattaac ctaggctgct gccaccgctg agcaataact
     1021 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aacctcaggc
     1081 atttgagaag cacacggtca cactgcttcc ggtagtcaat aaaccggtaa accagcaata
     1141 gacataagcg gctatttaac gaccctgccc tgaaccgacg accgggtcga atttgctttc
     1201 gaatttctgc cattcatccg cttattatca cttattcagg cgtagcaacc aggcgtttaa
     1261 gggcaccaat aactgcctta aaaaaattac gccccgccct gccactcatc gcagtactgt
     1321 tgtaattcat taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga
     1381 atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat agtgaaaacg
     1441 ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag
     1501 ggattggctg atacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt
     1561 tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg
     1621 tattcactcc agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg
     1681 tgaacactat cccatatcac cagctcaccg tctttcattg ccatacggaa ctccggatga
     1741 gcattcatca ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc
     1801 tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga
     1861 gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg
     1921 gtatatccag tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgataac
     1981 tcaaaaaata cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg
     2041 tgccgatcaa cgtctcattt tcgccaaaag ttggcccagg gcttcccggt atcaacaggg
     2101 acaccaggat ttatttattc tgcgaagtga tcttccgtca caggtattta ttcggcgcaa
     2161 agtgcgtcgg gtgatgctgc caacttactg atttagtgta tgatggtgtt tttgaggtgc
     2221 tccagtggct tctgtttcta tcagctgtcc ctcctgttca gctactgacg gggtggtgcg
     2281 taacggcaaa agcaccgccg gacatcagcg ctagcggagt gtatactggc ttactatgtt
     2341 ggcactgatg agggtgtcag tgaagtgctt catgtggcag gagaaaaaag gctgcaccgg
     2401 tgcgtcagca gaatatgtga tacaggatat attccgcttc ctcgctcact gactcgctac
     2461 gctcggtcgt tcgactgcgg cgagcggaaa tggcttacga acggggcgga gatttcctgg
     2521 aagatgccag gaagatactt aacagggaag tgagagggcc gcggcaaagc cgtttttcca
     2581 taggctccgc ccccctgaca agcatcacga aatctgacgc tcaaatcagt ggtggcgaaa
     2641 cccgacagga ctataaagat accaggcgtt tcccccttgc ggctccctcg tgcgctctcc
     2701 tgttcctgcc tttcggttta ccggtgtcat tccgctgtta tggccgcgtt tgtctcattc
     2761 cacgcctgac actcagttcc gggtaggcag ttcgctccaa gctggactgt atgcacgaac
     2821 cccccgttca gtccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     2881 aaagacatgc aaaagcacca ctggcagcag ccactggtaa ttgatttaga ggagttagtc
     2941 ttgaagtcat gcgccggtta aggctaaact gaaaggacaa gttttggtga ctgcgctcct
     3001 ccaagccagt tacctcggtt caaagagttg gtagctcaga gaaccttcga aaaaccgccc
     3061 tgcaaggcgg ttttttcgtt ttcagagcaa gagattacgc gcagaccaaa acgatctcaa
     3121 gaagatcatc ttattaatca gataaaatat ttctagattt cagtgcaatt tatctcttca
     3181 aatgtagcac ctgaagtcag ccccatacga tataagttgt aattctcatg ttagtcatgc
     3241 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     3301 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     3361 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3421 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     3481 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     3541 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     3601 gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg
     3661 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     3721 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     3781 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     3841 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     3901 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     3961 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     4021 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     4081 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     4141 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     4201 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     4261 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     4321 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     4381 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     4441 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     4501 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaaat taatacgact
     4561 cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt tgtttaactt
     4621 taataatgag atataccatg atcagtctga ttgcggcgtt agcggtagat cgcgttatcg
     4681 gcatggaaaa cgccatgccg tggaacctgc ctgccgatct cgcctggttt aaacgcaaca
     4741 ccttaaataa acccgtgatt atgggccgcc atacctggga atcaatcggt cgtccgttgc
     4801 caggacgcaa aaatattatc ctcagcagtc aaccgggtac ggacgatcgc gtaacgtggg
     4861 tgaagtcggt ggatgaagcc atcgcggcgt gtggtgacgt accagaaatc atggtgattg
     4921 gcggcggtcg cgtttatgaa cagttcttgc caaaagcgca aaaactgtat ctgacgcata
     4981 tcgacgcaga agtggaaggc gacacccatt tcccggatta cgagccggat gactgggaat
     5041 cggtattcag cgaattccac gatgctgatg cgcagaactc tcacagctat tgctttgaga
     5101 ttctggagcg gcgggaacag gcgtcgacaa gcgactacaa agacgatgac gacaagtaac
     5161 ggccgcataa tgcttaagtc gaacagaaag taatcgtatt gtaca
//
