LOCUS       sequence-181353-        6220 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the full length DHFR Mm in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6220)
  AUTHORS   Levin-Kravets O, Tanner N, Shohat N, Attali I, Keren-Kaplan T, 
            Shusterman A, Artzi S, Varvak A, Reshef Y, Shi X, Zucker O, Baram T,
            Katina C, Pilzer I, Ben-Aroya S, Prag G
  TITLE     A bacterial genetic selection system for ubiquitylation cascade 
            discovery.
  JOURNAL   Nat Methods. 2016 Nov;13(11):945-952. doi: 10.1038/nmeth.4003. Epub 
            2016 Oct 3.
  PUBMED    27694912
REFERENCE   2  (bases 1 to 6220)
  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: 
            "10.1038/nmeth.4003"; journalName: "Nat Methods"; date: "2016-11"; 
            volume: "13"; issue: "11"; pages: "945-952"
COMMENT     Sequence Label: pGST-DHFR Mm
FEATURES             Location/Qualifiers
     source          1..6220
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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)."
     CDS             258..818
                     /codon_start=1
                     /gene="Mus musculus Dhfr"
                     /product="mouse dihydrofolate reductase"
                     /label=DHFR
                     /translation="MVRPLNCIVAVSQNMGIGKNGDLPWPPLRNEFKYFQRMTTTSSVE
                     GKQNLVIMGRKTWFSIPEKNRPLKDRINIVLSRELKEPPRGAHFLAKSLDDALRLIEQP
                     ELASKVDMVWIVGGSSVYQEAMNQPGHLRLFVTRIMQEFESDTFFPEIDLGKYKLLPEY
                     PGVLSEVQEEKGIKYKFEVYEKKD"
     CDS             complement(820..843)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             849..1409
                     /codon_start=1
                     /gene="Mus musculus Dhfr"
                     /product="mouse dihydrofolate reductase"
                     /label=DHFR
                     /translation="MVRPLNCIVAVSQNMGIGKNGDLPWPPLRNEFKYFQRMTTTSSVE
                     GKQNLVIMGRKTWFSIPEKNRPLKDRINIVLSRELKEPPRGAHFLAKSLDDALRLIEQP
                     ELASKVDMVWIVGGSSVYQEAMNQPGHLRLFVTRIMQEFESDTFFPEIDLGKYKLLPEY
                     PGVLSEVQEEKGIKYKFEVYEKKD"
     CDS             complement(1411..1434)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             1440..2093
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2051..2073
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2106..2126
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2270..2292)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2437..2455)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2523..2627
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2628..2696,2697..3488)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2846..2865)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3659..4247
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4148..4167
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4401..4418
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4491..4568
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4569..5651
                     /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(4588..4607)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5700..5705,5706..5723,5724..5730)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5738..5754
                     /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     5762..5778
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5762..5778
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5774..6220,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5794..5811)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5794..5810)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5803..5825)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 agcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtacatatg gttcgaccat tgaactgcat cgtcgccgtg tcccaaaata
      301 tggggattgg caagaacgga gacctaccct ggcctccgct caggaacgag ttcaagtact
      361 tccaaagaat gaccacaacc tcttcagtgg aaggtaaaca gaatctggtg attatgggta
      421 ggaaaacctg gttctccatt cctgagaaga atcgaccttt aaaggacaga attaatatag
      481 ttctcagtag agaactcaaa gaaccaccac gaggagctca ttttcttgcc aaaagtttgg
      541 atgatgcctt aagacttatt gaacaaccgg aattggcaag taaagtagac atggtttgga
      601 tagtcggagg cagttctgtt taccaggaag ccatgaatca accaggccac ctcagactct
      661 ttgtgacaag gatcatgcag gaatttgaaa gtgacacgtt tttcccagaa attgatttgg
      721 ggaaatataa acttctccca gaatacccag gcgtcctctc tgaggtccag gaggaaaaag
      781 gcatcaagta taagtttgaa gtctacgaga agaaagacgg tggtggtggt ggtggtggtg
      841 gtggtcatat ggttcgacca ttgaactgca tcgtcgccgt gtcccaaaat atggggattg
      901 gcaagaacgg agacctaccc tggcctccgc tcaggaacga gttcaagtac ttccaaagaa
      961 tgaccacaac ctcttcagtg gaaggtaaac agaatctggt gattatgggt aggaaaacct
     1021 ggttctccat tcctgagaag aatcgacctt taaaggacag aattaatata gttctcagta
     1081 gagaactcaa agaaccacca cgaggagctc attttcttgc caaaagtttg gatgatgcct
     1141 taagacttat tgaacaaccg gaattggcaa gtaaagtaga catggtttgg atagtcggag
     1201 gcagttctgt ttaccaggaa gccatgaatc aaccaggcca cctcagactc tttgtgacaa
     1261 ggatcatgca ggaatttgaa agtgacacgt ttttcccaga aattgatttg gggaaatata
     1321 aacttctccc agaataccca ggcgtcctct ctgaggtcca ggaggaaaaa ggcatcaagt
     1381 ataagtttga agtctacgag aagaaagacg gtggtggtgg tggtggtggt ggtggtcata
     1441 tgtcccctat actaggttat tggaaaatta agggccttgt gcaacccact cgacttcttt
     1501 tggaatatct tgaagaaaaa tatgaagagc atttgtatga gcgcgatgaa ggtgataaat
     1561 ggcgaaacaa aaagtttgaa ttgggtttgg agtttcccaa tcttccttat tatattgatg
     1621 gtgatgttaa attaacacag tctatggcca tcatacgtta tatagctgac aagcacaaca
     1681 tgttgggtgg ttgtccaaaa gagcgtgcag agatttcaat gcttgaagga gcggttttgg
     1741 atattagata cggtgtttcg agaattgcat atagtaaaga ctttgaaact ctcaaagttg
     1801 attttcttag caagctacct gaaatgctga aaatgttcga agatcgttta tgtcataaaa
     1861 catatttaaa tggtgatcat gtaacccatc ctgacttcat gttgtatgac gctcttgatg
     1921 ttgttttata catggaccca atgtgcctgg atgcgttccc aaaattagtt tgttttaaaa
     1981 aacgtattga agctatccca caaattgata agtacttgaa atccagcaag tatatagcat
     2041 ggcctttgca gggctggcaa gccacgtttg gtggtggcga ccatcctcca aaaatcgata
     2101 cgaccgaaaa cctgtatttt cagggcgcca tgggatccgg aattcaaagg cctacgtcga
     2161 cgagctcact agtcgcggcc gctttcgaat ctagagcctg cagtctcgag cggccgcatc
     2221 gtgactgact gacgatctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     2281 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     2341 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta
     2401 gcgatagcgg agtgtataat tcttgaagac gaaagggcct cgtgatacgc ctatttttat
     2461 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     2521 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     2581 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     2641 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     2701 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     2761 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     2821 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg
     2881 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     2941 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     3001 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     3061 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     3121 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     3181 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     3241 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     3301 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     3361 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     3421 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     3481 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     3541 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     3601 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3661 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     3721 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     3781 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     3841 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3901 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3961 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4021 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4081 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4141 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4201 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4261 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     4321 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     4381 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     4441 ggtattttct ccttacgcat ctgtgcggta tttcacaccg cataaattcc gacaccatcg
     4501 aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg
     4561 tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc
     4621 agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag
     4681 tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg
     4741 gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc
     4801 aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga
     4861 tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac
     4921 gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag
     4981 ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca
     5041 gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg
     5101 gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc
     5161 tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag
     5221 gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg
     5281 ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta
     5341 ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag
     5401 acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc
     5461 aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc
     5521 tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct
     5581 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     5641 gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc accccaggct
     5701 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     5761 acaggaaaca gctatgacca tgattacgga ttcactggcc gtcgttttac aacgtcgtga
     5821 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     5881 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     5941 tggcgaatgg cgctttgcct ggtttccggc accagaagcg gtgccggaaa gctggctgga
     6001 gtgcgatctt cctgaggccg atactgtcgt cgtcccctca aactggcaga tgcacggtta
     6061 cgatgcgccc atctacacca acgtgaccta tcccattacg gtcaatccgc cgtttgttcc
     6121 cacggagaat ccgacgggtt gttactcgct cacatttaat gttgatgaaa gctggctaca
     6181 ggaaggccag acgcgaatta tttttgatgg cgttggaatt
//
