LOCUS       sequence-212178-        5807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of E.coli DHFR N23PP/S148A mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5807)
  AUTHORS   Bhabha G, Lee J, Ekiert DC, Gam J, Wilson IA, Dyson HJ, Benkovic SJ,
            Wright PE
  TITLE     A dynamic knockout reveals that conformational fluctuations 
            influence the chemical step of enzyme catalysis.
  JOURNAL   Science. 2011 Apr 8;332(6026):234-8. doi: 10.1126/science.1198542.
  PUBMED    21474759
REFERENCE   2  (bases 1 to 5807)
  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: "10.1126/science"; 
            journalName: "Science"; date: "2011-04-8- 8"; volume: "332"; issue: 
            "6026"; pages: "234-8"
COMMENT     Sequence Label: pET27b-ecDHFR-N23PP/S148A
FEATURES             Location/Qualifiers
     source          1..5807
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(164..196)
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     RBS             748..770
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    785..809
                     /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)."
     primer_bind     complement(809..828)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(810..828)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(912..931)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1137..1214
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1215..2297
                     /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     complement(1234..1253)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3106..3297
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3291..3313)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3399..3538
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3454..3473
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3553..3570)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3724..4312)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3804..3823)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4423..4442)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4434..5249
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4434..5249
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4507..4526)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5341..5796)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5478..5499)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5690..5709
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gacatcctcg gggtcttccg
      181 gggcgagttc tggctggcta gcccgtttga tctcgagtgc ggccgcaagc ttgtcgacgg
      241 agctcgaatt cggatcctta acgacgctcg aggatttcga aacaatagct atgcgcgttc
      301 tgcgcatcag catcgtggaa ttcgctgaat accgattccc agtcatccgg ctcgtaatcc
      361 ggaaaatggg tgtcgccttc cacttctgca tcgatatgcg tcagatacag tttttgcgct
      421 tttggcaaga actgttcata aacgcgtccg ccgccaatca ccatgatttc tggtacgtca
      481 ccacacgccg cgatggcttc gtcgaccgat ttaacccagg taacccgatc atcggtcccg
      541 ggttgactgc tgaggataat atttttgcgg ccgggcaaag gcctaccgat tgattcccag
      601 gtatggcgcc ccataatcac gggtttattt aaggtgttgc gtttaaacca ggcgagatcg
      661 gcaggcagcg gcggccatgg catggcgttt tccatgccga taacgcgatc taccgctagc
      721 gccgcaatca gactgatcat atgtatatct ccttcttaaa gttaaacaaa attatttcta
      781 gaggggaatt gttatccgct cacaattccc ctatagtgag tcgtattaat ttcgcgggat
      841 cgagatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc
      901 ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
      961 gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt
     1021 gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct
     1081 actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag atcccggaca
     1141 ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     1201 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
     1261 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
     1321 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     1381 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
     1441 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     1501 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     1561 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     1621 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     1681 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
     1741 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     1801 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
     1861 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
     1921 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
     1981 ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata
     2041 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     2101 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     2161 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
     2221 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     2281 tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg
     2341 ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg
     2401 ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag
     2461 gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag cgcgacgatg
     2521 atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc cttcgtcact
     2581 ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat ggcggcccca
     2641 cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt
     2701 actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa
     2761 acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg
     2821 gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc
     2881 tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg
     2941 atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt
     3001 aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt
     3061 atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa
     3121 aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct tctggagaaa
     3181 ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct
     3241 gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     3301 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     3361 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt
     3421 agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag
     3481 tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     3541 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3601 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3661 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3721 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3781 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3841 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3901 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3961 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4021 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4081 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4141 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     4201 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4261 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4321 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4381 ggtcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc
     4441 atattcaacg ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg gatgctgatt
     4501 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat
     4561 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca
     4621 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga
     4681 ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg
     4741 ggaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg
     4801 cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca
     4861 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg
     4921 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc
     4981 ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata
     5041 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg
     5101 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat
     5161 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt
     5221 ttcatttgat gctcgatgag tttttctaag aattaattca tgagcggata catatttgaa
     5281 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5341 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     5401 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     5461 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     5521 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     5581 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     5641 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     5701 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     5761 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgcca
//
