LOCUS       sequence-379259-        6093 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human dihydrofolate reductase (DHFR) with C-terminal 
            AviTag (Bio) and His10 sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6093)
  AUTHORS   Rana S, Dranchak P, Dahlin JL, Lamy L, Li W, Oliphant E, Shrimp JH, 
            Rajacharya GH, Tharakan R, Holland DO, Whitten AS, Wilson KM, Singh 
            PK, Durum SK, Tao D, Rai G, Inglese J
  TITLE     Methotrexate-based PROTACs as DHFR-specific chemical probes.
  JOURNAL   Cell Chem Biol. 2024 Feb 15;31(2):221-233.e14. doi: 
            10.1016/j.chembiol.2023.09.014. Epub 2023 Oct 23.
  PUBMED    37875111
REFERENCE   2  (bases 1 to 6093)
  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: 
            "10.1016/j.chembiol.2023.09.014"; journalName: "Cell Chem Biol"; 
            date: "2024-02-15- 15"; volume: "31"; issue: "2"; pages: 
            "221-233.e14"
COMMENT     Sequence Label: pET21a HsDHFR-bio-10His
FEATURES             Location/Qualifiers
     source          1..6093
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1362..1553
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1547..1569)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1655..1797
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1710..1729
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1812..1829)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1983..2571)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2063..2082)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2742..3533,3534..3602))
                     /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     3365..3384
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3603..3707)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3734..4189)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3871..3892)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4083..4102
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4226..4273)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4269..4287
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4340..4357)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4385..4414)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             complement(4424..4441)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(4442..4486)
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             complement(5057..5089)
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     RBS             5097..5119
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5134..5158
                     /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(5158..5177)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5159..5177)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5261..5280)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5486..5563
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5564..6093,1..553)
                     /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(5583..5602)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg
       61 tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga
      121 aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat
      181 cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat
      241 taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga
      301 agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg
      361 gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca
      421 gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc
      481 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
      541 aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat
      601 ctcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca
      661 tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc
      721 cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg
      781 gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc
      841 ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccccacggg
      901 tgcgcatgat cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg
      961 gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt
     1021 ctgcgacctg agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa
     1081 cgcggaagtc agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc
     1141 taccctgtgg aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt
     1201 ttctctggtc ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc
     1261 gggcatgttc atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca
     1321 ttacccccat gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa
     1381 ccgcccttaa catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca
     1441 acgagctgga cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg
     1501 agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     1561 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     1621 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     1681 atagcggagt gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     1741 ccatatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
     1801 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     1861 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     1921 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1981 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     2041 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2101 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2161 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     2221 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2281 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2341 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2401 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     2461 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2521 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2581 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2641 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2701 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     2761 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     2821 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     2881 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     2941 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3001 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca
     3061 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3121 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3181 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3241 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3301 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3361 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3421 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3481 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3541 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     3601 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3661 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     3721 aaagtgccac ctgaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt
     3781 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     3841 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     3901 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     3961 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     4021 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     4081 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     4141 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
     4201 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
     4261 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
     4321 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagctttt
     4381 attagtggtg gtggtgatgg tgatggtggt ggtggccagc cgcagaacca cgcggaacca
     4441 gttcgtgcca ttcaattttc tgcgcttcga agatatcgtt cagaccatcg tttttctcat
     4501 aaacttcgaa tttgtattta atgccttttt cttcctgaac atcagacaga acgcccgggt
     4561 attccggcag cagtttgtat ttttccagat cgatttccgg gaagaaggta tcgctttcga
     4621 aatcctgcat gatacgggta acgaacagtt tcaggtggcc cgggtggttc atcgcttctt
     4681 tgtaaacgct gctaccacca acgatccaaa ccatatcaac tttgttcgcc agttccggct
     4741 gttcggtcag tttcagagca tcatccagag aacggctcag gaagtgcgca ccctgcggcg
     4801 gttctttcag ttcacgagac agaaccaggt tgatacgacc tttcagcgga cggtttttct
     4861 ccgggataga gaaccaggtc tttttaccca tgataaccag gttctgtttg ccttcaacag
     4921 agctggtggt ggtcatacgc tggaagtaac ggaattcgtt acgcagcggc ggccacggca
     4981 gatcgccgtt tttaccgatg cccatgttct gagaaaccgc aacgatgcag ttcagagaac
     5041 caaccatgga tccgcgaccc atttgctgtc caccagtcat gctagccata tgtatatctc
     5101 cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc
     5161 tatagtgagt cgtattaatt tcgcgggatc gagatctcga tcctctacgc cggacgcatc
     5221 gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc
     5281 gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg
     5341 gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt
     5401 gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg
     5461 cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat
     5521 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt
     5581 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca
     5641 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa
     5701 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt
     5761 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg
     5821 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc
     5881 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta
     5941 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt
     6001 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg
     6061 tacgcgactg ggcgtggagc atctggtcgc att
//
