LOCUS       sequence-229500-        5895 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of E.coli DHFR N23PP, L28F, and G51PEKN mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5895)
  AUTHORS   Liu CT, Hanoian P, French JB, Pringle TH, Hammes-Schiffer S, 
            Benkovic SJ
  TITLE     Functional significance of evolving protein sequence in 
            dihydrofolate reductase from bacteria to humans.
  JOURNAL   Proc Natl Acad Sci U S A. 2013 Jun 18;110(25):10159-64. doi: 
            10.1073/pnas.1307130110. Epub 2013 Jun 3.
  PUBMED    23733948
REFERENCE   2  (bases 1 to 5895)
  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: 
            "10.1073/pnas.1307130110"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2013-06-18- 18"; volume: "110"; issue: "25"; pages: 
            "10159-64"
COMMENT     Sequence Label: pET22b-ecDHFR-N23PP/L28F/G51PEKN
FEATURES             Location/Qualifiers
     source          1..5895
                     /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"
     RBS             703..725
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    740..764
                     /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(764..783)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(765..783)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(867..886)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1092..1169
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1170..2252
                     /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(1189..1208)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3061..3252
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3246..3268)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3354..3493
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3409..3428
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3508..3525)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3679..4267)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3759..3778)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4438..5229,5230..5298))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5061..5080
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5299..5403)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5429..5884)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5566..5587)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5778..5797
                     /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 gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccttaacga cgctcgagga tttcgaaaca atagctatgc
      241 gagttctgcg catcagcatc gtggaattcg ctgaataccg attcccagtc atccggctcg
      301 taatccggaa aatgggtgtc gccttccact tctgcatcga tatgcgtcag atacagtttt
      361 tgcgcttttg gcaagaactg ttcataaacg cgtccgccgc caatcaccat gatttctggt
      421 acgtcaccac acgccgcgat ggcttcgtcg accgatttaa cccaggtaac ccgatcatcg
      481 gtcccgggtt gactgctgag gataatattt ttgcggccgg gcaaaggcct gtttttttct
      541 gggattgatt cccaggtatg gcgccccata atcacgggtt tatttaaggt gttgcgttta
      601 aaccaggcga aatcggcagg cagcggcggc catggcatgg cgttttccat gccgataacg
      661 cgatctaccg ctagcgccgc aatcagactg atcatatgta tatctccttc ttaaagttaa
      721 acaaaattat ttctagaggg gaattgttat ccgctcacaa ttcccctata gtgagtcgta
      781 ttaatttcgc gggatcgaga tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac
      841 cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg
      901 ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt
      961 ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct
     1021 caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg
     1081 tcgagatccc ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc
     1141 cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag
     1201 agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt
     1261 ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc
     1321 gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc
     1381 tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg
     1441 gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg
     1501 tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc
     1561 aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct
     1621 ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg
     1681 tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt
     1741 ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc
     1801 agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc
     1861 aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc
     1921 tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag
     1981 tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac
     2041 aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc
     2101 aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg
     2161 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     2221 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc
     2281 actcattagg caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc
     2341 ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg
     2401 caactcgtag gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc
     2461 tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct
     2521 caagccttcg tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc
     2581 ggcatggcgg ccccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc
     2641 tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc
     2701 gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt
     2761 gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc
     2821 atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg
     2881 cattgaccct gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc
     2941 tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc
     3001 tctcgtttca tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca
     3061 gtgaccaaac aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta
     3121 acgcttctgg agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg
     3181 cttcacgacc acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt
     3241 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     3301 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
     3361 atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc
     3421 agattgtact gagagtgcac cattgcggtg tgaaataccg cacagatgcg taaggagaaa
     3481 ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     3541 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3601 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3661 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3721 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3781 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3841 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3901 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3961 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4021 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4081 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     4141 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4201 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     4261 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4321 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4381 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4441 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4501 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4561 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4621 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4681 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4741 tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4801 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     4861 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     4921 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     4981 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     5041 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5101 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     5161 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     5221 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     5281 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     5341 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5401 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
     5461 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     5521 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     5581 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     5641 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     5701 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     5761 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     5821 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     5881 gcgtcccatt cgcca
//
