LOCUS       sequence-163060-        7879 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Residues 1-702 of ALIX; Bacterial Expression Vector; TEV cleavable 
            N-term His-tag; Sundquist Lab Internal ID: WISP10-648.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7879)
  AUTHORS   McCullough J, Fisher RD, Whitby FG, Sundquist WI, Hill CP
  TITLE     ALIX-CHMP4 interactions in the human ESCRT pathway.
  JOURNAL   Proc Natl Acad Sci U S A. 2008 Jun 3;105(22):7687-91. doi: 
            10.1073/pnas.0801567105. Epub 2008 May 29.
  PUBMED    18511562
REFERENCE   2  (bases 1 to 7879)
  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: 
            "10.1073/pnas.0801567105"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2008-06-3- 3"; volume: "105"; issue: "22"; pages: "7687-91"
COMMENT     Sequence Label: pet151D-TOPO ALIX Bro1-V (1-702)
FEATURES             Location/Qualifiers
     source          1..7879
                     /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(2580..2598)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2594..2641
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2879..2897)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2965..3069
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3070..3138,3139..3930)
                     /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(3288..3307)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4101..4689
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4590..4609
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4843..4860
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4875..5014
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4940..4959)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5100..5122
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5116..5307)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6619..7701)
                     /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     7663..7682
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7702..7779)
                     /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 tcacccatat ggcgacattc atctcggtgc
      421 agctgaaaaa gacctcagag gtggacctgg ccaagccgct ggtgaagttc atccagcaga
      481 cttacccaag cggcggggaa gagcaggccc agtactgccg cgcggcggag gagctcagca
      541 agctgcgccg cgccgcagtc ggtcgtccgc tggacaagca cgagggcgcg ctcgagacgc
      601 tcctgagata ttatgatcag atttgttcta ttgaacccaa attcccattt tctgaaaatc
      661 agatctgctt gacatttacc tggaaggatg ctttcgataa aggttcactt tttggaggct
      721 ctgtaaaact ggctcttgca agcttaggat atgaaaagag ctgtgtgttg ttcaattgtg
      781 cagccttagc tagccaaatt gcagcagaac agaacctgga taatgatgaa ggattgaaaa
      841 tcgctgctaa acattaccag tttgctagtg gtgccttttt acatattaaa gagacggttt
      901 tatctgcctt aagtcgagag ccgaccgtgg acatatctcc agatactgtt gggaccctca
      961 gtcttattat gctggcacag gctcaagaag tatttttttt aaaagccaca agagataaaa
     1021 tgaaagatgc catcatagct aaattggcta atcaggctgc agattatttt ggtgatgctt
     1081 tcaaacagtg tcaatacaaa gatactctcc ccaaggaggt gttccctgtc ttggctgcaa
     1141 agcactgtat catgcaggcc aatgctgagt accatcagtc tatcctggca aaacagcaga
     1201 agaaatttgg agaagaaatt gcaaggttac agcatgcagc agaactgatt aaaacagtgg
     1261 catctcgcta tgatgaatat gttaatgtga aggatttttc tgacaaaatc aatcgtgccc
     1321 ttgctgcagc aaagaaggat aatgacttca tttatcatga tcgagttcca gaccttaaag
     1381 atctagatcc tattggcaaa gccacacttg tgaaatctac cccggtcaat gtacccatca
     1441 gtcagaaatt tactgatctg tttgagaaga tggttcccgt gtcagtacag cagtctttgg
     1501 ctgcctataa tcagaggaaa gccgatttgg ttaacagatc aattgctcag atgagagaag
     1561 ccaccacttt ggcaaatggg gtgctagctt cccttaatct tccagcagca attgaagatg
     1621 tgtctggaga cactgtacct cagtctatat tgactaaatc cagatctgtg attgaacagg
     1681 gaggcatcca gactgttgat cagttgatta aagaactgcc tgaattactg caacgaaata
     1741 gagaaatcct agatgagtca ttaaggttgt tggatgaaga agaagcaacc gataatgatt
     1801 taagagcaaa atttaaggaa cgttggcaaa ggacaccatc caatgaactg tataagcctt
     1861 taagagcaga gggaaccaac ttcagaacag ttttagataa agctgtgcag gcagatggac
     1921 aagtgaaaga atgttaccag tctcatcgtg acaccatcgt gcttttgtgt aagccagagc
     1981 ctgagctgaa tgctgccatc ccttctgcta atccagcaaa gaccatgcag ggcagtgagg
     2041 ttgtaaatgt cttaaaatcc ttattgtcaa atcttgatga agtaaagaag gaaagagagg
     2101 gtctggagaa tgacttgaaa tctgtgaatt ttgacatgac aagcaagttt ttgacagccc
     2161 tggctcaaga tggtgtgata aatgaagaag ctctttctgt tactgaacta gatcgagtct
     2221 atggaggtct tacaactaaa gtccaagaat ctctaaagaa acaggaggga cttcttaaaa
     2281 atattcaggt ctcacatcag gaattttcaa aaatgaaaca atctaataat gaagctaact
     2341 taagagaaga agttttgaag aatttagcta ctgcgtatga caactttgtt gaacttgtag
     2401 ctaatttgaa ggaaggcaca aagttttaca atgagttgac tgaaatcctg gtcaggttcc
     2461 agaacaaatg cagtgatata gtttttgcac ggaagacaga aagataagga tccaattaag
     2521 ggcgagctca gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     2581 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     2641 gctgaaagga ggaactatat ccggatatcc cgcaagaggc ccggcagtac cggcataacc
     2701 aagcctatgc ctacagcatc cagggtgacg gtgccgagga tgacgatgag cgcattgtta
     2761 gatttcatac acggtgcctg actgcgttag caatttaact gtgataaact accgcattaa
     2821 agctagctta tcgatgataa gctgtcaaac atgagaatta attcttgaag acgaaagggc
     2881 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     2941 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     3001 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     3061 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     3121 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     3181 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     3241 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     3301 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     3361 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     3421 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     3481 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     3541 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     3601 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3661 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3721 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     3781 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     3841 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3901 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     3961 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     4021 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     4081 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4141 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4201 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     4261 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4321 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4381 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4441 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4501 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4561 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4621 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4681 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4741 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4801 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4861 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4921 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     4981 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     5041 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     5101 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     5161 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     5221 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     5281 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     5341 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     5401 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     5461 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     5521 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     5581 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     5641 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     5701 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     5761 catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga
     5821 gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg
     5881 caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct
     5941 tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca
     6001 aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca
     6061 taaatcgccg tgacgatcag cggtccaatg atcgaagtta ggctggtaag agccgcgagc
     6121 gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat ggcctgcaac
     6181 gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc catccagcct
     6241 cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg
     6301 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     6361 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     6421 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     6481 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     6541 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     6601 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     6661 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     6721 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     6781 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     6841 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     6901 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     6961 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     7021 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     7081 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     7141 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     7201 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     7261 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     7321 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     7381 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     7441 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     7501 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     7561 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     7621 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     7681 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     7741 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     7801 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     7861 ccgcaaggaa tggtgcatg
//
