LOCUS       sequence-209085-        5883 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of E.coli DHFR protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5883)
  AUTHORS   Cameron CE, Benkovic SJ
  TITLE     Evidence for a functional role of the dynamics of glycine-121 of 
            Escherichia coli dihydrofolate reductase obtained from kinetic 
            analysis of a site-directed mutant.
  JOURNAL   Biochemistry. 1997 Dec 16;36(50):15792-800. doi: 10.1021/bi9716231.
  PUBMED    9398309
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"; journalName: 
            "Biochemistry"; date: "1997-12-16- 16"; volume: "36"; issue: "50"; 
            pages: "15792-800"
COMMENT     Sequence Label: pET22b-ecDHFR
FEATURES             Location/Qualifiers
     source          1..5883
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(33..1115)
                     /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     1077..1096
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1116..1193)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1399..1418
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1502..1521
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1502..1520
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1521..1545
                     /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)."
     RBS             1560..1582
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2116..2133
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2186..2204)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2200..2247
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2284..2739
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2371..2390)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2581..2602
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2765..2869
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2870..2938,2939..3730)
                     /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     complement(3088..3107)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3901..4489
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4390..4409
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4643..4660
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4675..4814
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4740..4759)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4900..4922
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4916..5107)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
       61 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
      121 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
      181 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
      241 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
      301 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
      361 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
      421 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
      481 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
      541 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
      601 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
      661 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
      721 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
      781 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
      841 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
      901 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
      961 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     1021 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     1081 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     1141 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     1201 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     1261 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     1321 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     1381 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     1441 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa
     1501 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt
     1561 ttgtttaact ttaagaagga gatatacata tgatcagtct gattgcggcg ctagcggtag
     1621 atcgcgttat cggcatggaa aacgccatgc catggaacct gcctgccgat ctcgcctggt
     1681 ttaaacgcaa caccttaaat aaacccgtga ttatggggcg ccatacctgg gaatcaatcg
     1741 gtaggccttt gcccggccgc aaaaatatta tcctcagcag tcaacccggg accgatgatc
     1801 gggttacctg ggttaaatcg gtcgacgaag ccatcgcggc gtgtggtgac gtaccagaaa
     1861 tcatggtgat tggcggcgga cgcgtttatg aacagttctt gccaaaagcg caaaaactgt
     1921 atctgacgca tatcgatgca gaagtggaag gcgacaccca ttttccggat tacgagccgg
     1981 atgactggga atcggtattc agcgaattcc acgatgctga tgcgcagaac tcgcatagct
     2041 attgtttcga aatcctcgag cgtcgttaag gatccgaatt cgagctccgt cgacaagctt
     2101 gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     2161 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     2221 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     2281 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2341 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2401 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2461 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2521 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2581 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2641 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     2701 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     2761 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2821 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     2881 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     2941 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     3001 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     3061 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     3121 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     3181 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     3241 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     3301 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     3361 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat
     3421 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     3481 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     3541 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     3601 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     3661 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     3721 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     3781 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     3841 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3901 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3961 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4021 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     4081 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4141 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4201 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4261 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4321 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4381 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4441 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4501 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     4561 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     4621 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     4681 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     4741 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     4801 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     4861 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     4921 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     4981 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     5041 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     5101 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     5161 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     5221 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     5281 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     5341 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     5401 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     5461 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     5521 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     5581 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     5641 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     5701 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     5761 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     5821 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     5881 aat
//
