LOCUS       sequence-177422-        6999 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses full length human BROX with mutation L212D in bacteria; 
            internal ID WISP11-300.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6999)
  AUTHORS   Zhai Q, Landesman MB, Robinson H, Sundquist WI, Hill CP
  TITLE     Structure of the Bro1 domain protein BROX and functional analyses of
            the ALIX Bro1 domain in HIV-1 budding.
  JOURNAL   PLoS One. 2011;6(12):e27466. doi: 10.1371/journal.pone.0027466. Epub
            2011 Dec 5.
  PUBMED    22162750
REFERENCE   2  (bases 1 to 6999)
  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: "PLoS One.";
            date: "2011"; volume: "6(12):e27466. doi"; pages: " 
            10.1371/journal.pone.0027466. Epub 2011 Dec 5"
COMMENT     Sequence Label: pet151D-TOPO-BROX 1-411; L212D
FEATURES             Location/Qualifiers
     source          1..6999
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             300..317
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             318..359
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             360..380
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1700..1718)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1714..1761
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1999..2017)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2085..2189
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2190..2258,2259..3050)
                     /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(2408..2427)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3221..3809
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3710..3729
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3963..3980
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3995..4134
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4060..4079)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4220..4242
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4236..4427)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5739..6821)
                     /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     6783..6802
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6822..6899)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgc
      301 atcatcacca tcaccatggt aagcctatcc ctaaccctct cctcggtctc gattctacgg
      361 aaaacctgta ttttcaggga attgatccct tcaccatgac ccattggttt cataggaacc
      421 cattaaaagc cacagctcct gtgtctttta attactatgg tgtagtcact ggcccttctg
      481 cttcaaaaat atgcaatgac ttgaggtcat ccagggcacg actccttgaa ctgttcactg
      541 atttgagctg taatccagaa atgatgaaga atgcagcaga ttcatatttc tcacttttac
      601 aaggtttcat aaattctttg gatgaatcta cccaagaaag caagttacga tatattcaaa
      661 atttcaagtg gactgataca ttgcaaggac aggttccaag tgcccagcag gatgctgttt
      721 ttgaattaat ttccatggga tttaatgtag ctttatggta taccaaatat gcttcaagac
      781 tggctggaaa agaaaatata acagaagatg aagcaaaaga agttcatcga agcctaaaga
      841 ttgcagctgg gatttttaaa catttaaagg aaagtcatct cccaaaactc attacacctg
      901 cggaaaaagg aagagattta gagtcacgac tcatagaagc atacgttatt caatgtcagg
      961 ctgaagctca agaagtaaca attgctcgag caattgaact aaaacatgct cctggactaa
     1021 ttgctgcaga tgcgtatgaa acagccaatt tctatcaaaa agctgatcat actttatcca
     1081 gtttggagcc tgcatattct gccaaatgga gaaaatatct tcacttgaag atgtgtttct
     1141 acacagctta tgcttactgt taccatggtg agactttatt ggctagtgat aaatgcggtg
     1201 aagcaatcag gtctctccaa gaagcagaaa aattgtatgc aaaggcagaa gcactgtgta
     1261 aagaatatgg agaaaccaaa ggacctggac caacagtcaa accttcagga catctgttct
     1321 ttaggaaact tggaaacctt gtgaagaaca ccctagaaaa atgtcagaga gaaaatggat
     1381 ttatttactt tcaaaaaatt ccaacagaag ccccacagct ggaactcaaa gcaaattatg
     1441 gtctcgtaga gcctatacct ttcgaatttc ctcctacaag tgttcagtgg acaccagaaa
     1501 cattggctgc atttgatctc accaaaagac ccaaggatga cagtactaaa cccaaaccag
     1561 aagaagaagt gaaacctgtg aaagaaccag acatcaaacc tcaaaaggac actgggtgct
     1621 acatctccta acatatgaag ggcgagctca gatccggctg ctaacaaagc ccgaaaggaa
     1681 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     1741 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggatatcc cgcaagaggc
     1801 ccggcagtac cggcataacc aagcctatgc ctacagcatc cagggtgacg gtgccgagga
     1861 tgacgatgag cgcattgtta gatttcatac acggtgcctg actgcgttag caatttaact
     1921 atgataaact accgcattaa agctagctta tcgatgataa gctgtcaaac atgagaatta
     1981 attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     2041 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     2101 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     2161 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     2221 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     2281 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     2341 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     2401 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc
     2461 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     2521 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     2581 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     2641 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     2701 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
     2761 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     2821 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     2881 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     2941 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     3001 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     3061 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     3121 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3181 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3241 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     3301 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     3361 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     3421 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     3481 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     3541 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     3601 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     3661 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3721 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3781 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3841 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3901 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3961 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     4021 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     4081 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     4141 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     4201 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     4261 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     4321 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     4381 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     4441 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     4501 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     4561 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     4621 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     4681 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     4741 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     4801 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     4861 ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgagatg
     4921 cgccgcgtgc ggctgctgga gatggcggac gcgatggata tgttctgcca agggttggtt
     4981 tgcgcattca cagttctccg caagaattga ttggctccaa ttcttggagt ggtgaatccg
     5041 ttagcgaggt gtcgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac
     5101 gcaacgcggg gaggcagaca aggtataggg cggcgcctac aatccatgcc aacccgttcc
     5161 atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccaatg atcgaagtta
     5221 ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca tctacctgcc
     5281 tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa
     5341 tggggaaggc catccagcct cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg
     5401 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     5461 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     5521 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     5581 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     5641 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     5701 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5761 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5821 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     5881 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     5941 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     6001 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     6061 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     6121 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     6181 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     6241 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     6301 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     6361 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     6421 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     6481 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     6541 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     6601 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     6661 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     6721 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     6781 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     6841 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     6901 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     6961 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatg
//
