LOCUS       sequence-370140-        5969 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of human DHFR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5969)
  AUTHORS   Bhabha G, Tuttle L, Martinez-Yamout MA, Wright PE
  TITLE     Identification of endogenous ligands bound to bacterially expressed 
            human and E. coli dihydrofolate reductase by 2D NMR.
  JOURNAL   FEBS Lett. 2011 Nov 16;585(22):3528-32. Epub 2011 Oct 20.
  PUBMED    22024482
REFERENCE   2  (bases 1 to 5969)
  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"; journalName: "FEBS 
            Lett."; date: "2011-11-16"; volume: "585(22)"; pages: "3528-32. Epub
            2011 Oct 20"
COMMENT     Sequence Label: hDHFR
FEATURES             Location/Qualifiers
     source          1..5969
                     /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             777..799
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    814..838
                     /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(838..857)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(839..857)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(941..960)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1166..1243
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1244..2326
                     /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(1263..1282)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3135..3326
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3320..3342)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3428..3567
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3483..3502
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3582..3599)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3753..4341)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3833..3852)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4512..5303,5304..5372))
                     /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     5135..5154
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5373..5477)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5503..5958)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5640..5661)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5852..5871
                     /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 tcctattaat cattcttctc atatacttca aatttgtact
      241 taatgccttt ctcctcctgg acatcagaga gaacacctgg gtattctggc agaagtttat
      301 atttctccaa atcaatttct ggaaaaaacg tgtcactttc aaagtcttgc atgatccttg
      361 tcacaaatag tttaagatgg cctgggtgat tcatggcttc cttataaaca gaactgccac
      421 caactatcca gaccatgtct actttatttg ctaattctgg ttgttcagta agttttaagg
      481 catcatctag acttctggaa agaaaatgag ctccttgtgg aggttccttg agttctctgc
      541 tgagaactaa attaattcta ccctttaaag gtcgattctt ctcaggaatg gagaaccagg
      601 tcttcttacc cataatcacc agattctgtt taccttctac tgaagaggtt gtggtcattc
      661 tctggaaata tctgaattca ttcctgagcg gtggccaggg caggtccccg ttcttgccga
      721 tgcccatgtt ctgggacaca gcgacgatgc agtttagcga accaaccata tgtatatctc
      781 cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc
      841 tatagtgagt cgtattaatt tcgcgggatc gagatctcga tcctctacgc cggacgcatc
      901 gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc
      961 gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg
     1021 gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt
     1081 gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg
     1141 cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat
     1201 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt
     1261 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca
     1321 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa
     1381 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt
     1441 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg
     1501 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc
     1561 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta
     1621 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt
     1681 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg
     1741 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc
     1801 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac
     1861 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt
     1921 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa
     1981 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc
     2041 ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt
     2101 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca
     2161 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag
     2221 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     2281 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     2341 atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca
     2401 acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg
     2461 tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg
     2521 aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct
     2581 tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc
     2641 aggccattat cgccggcatg gcggccccac gggtgcgcat gatcgtgctc ctgtcgttga
     2701 ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg
     2761 agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg
     2821 tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta
     2881 tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat
     2941 taacgaagcg ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg
     3001 ccagttgttt accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta
     3061 tcgtgagcat cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt
     3121 acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca
     3181 gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag
     3241 acatctgtga atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt
     3301 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     3361 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     3421 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta
     3481 tgcggcatca gagcagattg tactgagagt gcaccattgc ggtgtgaaat accgcacaga
     3541 tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg
     3601 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3661 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3721 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3781 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3841 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     3901 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     3961 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4021 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4081 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4141 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     4201 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4261 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4321 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4381 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4441 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     4501 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4561 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4621 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4681 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4741 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4801 agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt
     4861 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     4921 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     4981 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5041 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5101 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5161 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5221 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5281 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5341 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     5401 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5461 caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata
     5521 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     5581 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     5641 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     5701 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     5761 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     5821 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     5881 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     5941 cgccgctaca gggcgcgtcc cattcgcca
//
