LOCUS       sequence-212223-        5883 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of E.coli DHFR C85A/C152S/T46C mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5883)
  AUTHORS   Liu CT, Layfield JP, Stewart RJ 3rd, French JB, Hanoian P, Asbury 
            JB, Hammes-Schiffer S, Benkovic SJ
  TITLE     Probing the electrostatics of active site microenvironments along 
            the catalytic cycle for Escherichia coli dihydrofolate reductase.
  JOURNAL   J Am Chem Soc. 2014 Jul 23;136(29):10349-60. doi: 10.1021/ja5038947.
            Epub 2014 Jul 11.
  PUBMED    24977791
REFERENCE   2  (bases 1 to 5883)
  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: "10.1021/ja5038947";
            journalName: "J Am Chem Soc"; date: "2014-07-23- 23"; volume: "136";
            issue: "29"; pages: "10349-60"
COMMENT     Sequence Label: pET22b-ecDHFR-dCys-T46C
FEATURES             Location/Qualifiers
     source          1..5883
                     /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             691..713
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    728..752
                     /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(752..771)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(753..771)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(855..874)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1080..1157
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1158..2240
                     /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(1177..1196)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3049..3240
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3234..3256)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3342..3481
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3397..3416
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3496..3513)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3667..4255)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3747..3766)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4426..5217,5218..5286))
                     /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     5049..5068
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5287..5391)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5417..5872)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5554..5575)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5766..5785
                     /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 tttcgaaact atagctatgc
      241 gagttctgcg catcagcatc gtggaattcg ctgaataccg attcccagtc atccggctcg
      301 taatccggaa aatgggtgtc gccttccact tctgcatcga tatgcgtcag atacagtttt
      361 tgcgcttttg gcaagaactg ttcataaacg cgtccgccgc caatcaccat gatttctggt
      421 acgtcaccag ccgccgcgat ggcttcgtcg accgatttaa cccaggtaac ccgatcatcg
      481 gtcccgggtt gactgctgag gataatattt ttgcggccgg gcaaaggcct accgattgat
      541 tcccagcaat ggcgccccat aatcacgggt ttatttaagg tgttgcgttt aaaccaggcg
      601 agatcggcag gcaggttcca tggcatggcg ttttccatgc cgataacgcg atctaccgct
      661 agcgccgcaa tcagactgat catatgtata tctccttctt aaagttaaac aaaattattt
      721 ctagagggga attgttatcc gctcacaatt cccctatagt gagtcgtatt aatttcgcgg
      781 gatcgagatc tcgatcctct acgccggacg catcgtggcc ggcatcaccg gcgccacagg
      841 tgcggttgct ggcgcctata tcgccgacat caccgatggg gaagatcggg ctcgccactt
      901 cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgggggact
      961 gttgggcgcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca acggcctcaa
     1021 cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc gagatcccgg
     1081 acaccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc
     1141 aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg
     1201 tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc
     1261 gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac
     1321 aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg
     1381 cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg
     1441 tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc
     1501 tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg
     1561 ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac
     1621 ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg
     1681 tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc
     1741 gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg
     1801 aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg
     1861 agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc
     1921 gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg
     1981 ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc
     2041 tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg
     2101 gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc
     2161 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     2221 gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca
     2281 ccgggatctc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg
     2341 cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga
     2401 caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg
     2461 atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca agccttcgtc
     2521 actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg catggcggcc
     2581 ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc
     2641 cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc
     2701 aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt
     2761 ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc
     2821 tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga
     2881 gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc
     2941 agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc
     3001 ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag
     3061 gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag
     3121 aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac
     3181 gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga
     3241 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     3301 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca
     3361 cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga
     3421 gagtgcacca ttgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     3481 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     3541 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     3601 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     3661 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     3721 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     3781 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3841 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     3901 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     3961 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4021 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4081 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     4141 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4201 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4261 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     4321 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     4381 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     4441 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4501 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4561 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     4621 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     4681 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     4741 tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     4801 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     4861 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     4921 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     4981 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5041 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     5101 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     5161 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     5221 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     5281 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     5341 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     5401 ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     5461 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     5521 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     5581 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     5641 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     5701 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     5761 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     5821 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg
     5881 cca
//
