LOCUS       sequence-229499-        5892 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of E.coli DHFR G51PEKN mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5892)
  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 5892)
  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-G51PEKN
FEATURES             Location/Qualifiers
     source          1..5892
                     /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             700..722
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    737..761
                     /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(761..780)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(762..780)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(864..883)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1089..1166
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1167..2249
                     /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(1186..1205)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3058..3249
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3243..3265)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3351..3490
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3406..3425
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3505..3522)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3676..4264)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3756..3775)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4435..5226,5227..5295))
                     /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     5058..5077
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5296..5400)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5426..5881)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5563..5584)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5775..5794
                     /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 gatcggcagg caggttccat ggcatggcgt tttccatgcc gataacgcga
      661 tctaccgcta gcgccgcaat cagactgatc atatgtatat ctccttctta aagttaaaca
      721 aaattatttc tagaggggaa ttgttatccg ctcacaattc ccctatagtg agtcgtatta
      781 atttcgcggg atcgagatct cgatcctcta cgccggacgc atcgtggccg gcatcaccgg
      841 cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc
      901 tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc
      961 cgggggactg ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa
     1021 cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg
     1081 agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg
     1141 aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt
     1201 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg
     1261 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg
     1321 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg
     1381 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg
     1441 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc
     1501 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg
     1561 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg
     1621 accagacacc catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg
     1681 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg
     1741 tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc
     1801 cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa
     1861 tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg
     1921 gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg
     1981 gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg
     2041 attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg
     2101 cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc
     2161 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     2221 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact
     2281 cattaggcac cgggatctcg accgatgccc ttgagagcct tcaacccagt cagctccttc
     2341 cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa
     2401 ctcgtaggac aggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg
     2461 agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa
     2521 gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc
     2581 atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg
     2641 cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac
     2701 tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt
     2761 tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc
     2821 gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat
     2881 tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca
     2941 caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct
     3001 cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg
     3061 accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg
     3121 cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt
     3181 cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa
     3241 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     3301 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
     3361 acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga
     3421 ttgtactgag agtgcaccat tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     3481 ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     3541 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3601 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3661 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3721 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3781 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     3841 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     3901 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     3961 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4021 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4081 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     4141 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4201 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4261 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4321 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     4381 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     4441 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     4501 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     4561 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     4621 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     4681 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     4741 tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     4801 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     4861 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     4921 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     4981 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5041 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5101 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     5161 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     5221 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     5281 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     5341 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     5401 cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg
     5461 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     5521 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     5581 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     5641 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     5701 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     5761 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     5821 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     5881 tcccattcgc ca
//
