LOCUS       sequence-161131-        6786 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Residues 1-359 of ALIX; Bacterial Expression Vector; N-term His-tag;
            Sundquist Lab Internal ID: WISP11-428.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6786)
  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 6786)
  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: pet16b ALIX Bro1 (1-359)
FEATURES             Location/Qualifiers
     source          1..6786
                     /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             302..331
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             338..358
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1492..1510)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1506..1553
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1728..1733,1734..1750,1751..1756))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1783..1801)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1869..1973
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1974..2042,2043..2834)
                     /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(2192..2211)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3005..3593
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3494..3513
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3747..3764
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3779..3921
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3847..3866)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4007..4029
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4023..4214)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5526..6608)
                     /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     6570..6589
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6609..6686)
                     /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 ataccatggg
      301 ccatcatcat catcatcatc atcatcatca tagcggcgaa aacctgtatt tccagggcca
      361 tatggcgaca ttcatctcgg tgcagctgaa aaagacctca gaggtggacc tggccaagcc
      421 gctggtgaag ttcatccagc agacttaccc aagcggcggg gaagagcagg cccagtactg
      481 ccgcgcggcg gaggagctca gcaagctgcg ccgcgccgca gtcggtcgtc cgctggacaa
      541 gcacgagggc gcgctcgaga cgctcctgag atattatgat cagatttgtt ctattgaacc
      601 caaattccca ttttctgaaa atcagatctg cttgacattt acctggaagg atgctttcga
      661 taaaggttca ctttttggag gctctgtaaa actggctctt gcaagcttag gatatgaaaa
      721 gagctgtgtg ttgttcaatt gtgcagcctt agctagccaa attgcagcag aacagaactt
      781 agacaacgat gaaggattga aaatcgctgc taaacattac cagtttgcta gtggtgcctt
      841 tttacatatt aaagagacgg ttttatctgc cttaagtcga gagccgaccg tggacatatc
      901 tccagatact gttgggaccc tcagtcttat tatgctggca caggctcaag aagtattttt
      961 tttaaaagcc acaagagata aaatgaaaga tgccatcata gctaaattgg ctaatcaggc
     1021 tgcagattat tttggtgatg ctttcaaaca gtgtcaatac aaagatactc tccccaagga
     1081 ggtgttccct gtcttggctg caaagcactg tatcatgcag gccaatgctg agtaccatca
     1141 gtctatcctg gcaaaacagc agaagaaatt tggagaagaa attgcaaggt tacagcatgc
     1201 agcagaactg attaaaacag tggcatctcg ctatgatgaa tatgttaatg tgaaggattt
     1261 ttctgacaaa atcaatcgtg cccttgctgc agcaaagaag gataatgact tcatttatca
     1321 tgatcgagtt ccagacctta aagatctaga tcctattggc aaagccacac ttgtgaaatc
     1381 taccccggtc aatgtaccca tcagtcagaa atttactgat ctgtttgaga agatggttta
     1441 aggatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     1501 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     1561 gaggaactat atccggatat cccgcaagag gcccggcagt accggcataa ccaagcctat
     1621 gcctacagca tccagggtga cggtgccgag gatgacgatg agcgcattgt tagatttcat
     1681 acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt aaagcttatc
     1741 gatgataagc tgtcaaacat gagaattctt gaagacgaaa gggcctcgtg atacgcctat
     1801 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     1861 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     1921 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     1981 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     2041 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     2101 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     2161 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg
     2221 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     2281 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     2341 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     2401 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     2461 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     2521 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     2581 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     2641 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     2701 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     2761 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     2821 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     2881 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     2941 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3001 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3061 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3121 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     3181 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     3241 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3301 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     3361 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     3421 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     3481 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     3541 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     3601 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     3661 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     3721 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     3781 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tatggtgcac
     3841 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     3901 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     3961 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4021 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     4081 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     4141 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     4201 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     4261 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     4321 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     4381 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     4441 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     4501 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     4561 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     4621 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     4681 ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc tgctggagat ggcggacgcg
     4741 atggatatgt tctgccaagg gttggtttgc gcattcacag ttctccgcaa gaattgattg
     4801 gctccaattc ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc attcaggtcg
     4861 aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg tatagggcgg
     4921 cgcctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa atcgccgtga
     4981 cgatcagcgg tccagtgatc gaagttaggc tggtaagagc cgcgagcgat ccttgaagct
     5041 gtccctgatg gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg ggcatcccga
     5101 tgccgccgga agcgagaaga atcataatgg ggaaggccat ccagcctcgc gtcgcgaacg
     5161 ccagcaagac gtagcccagc gcgtcggccg ccatgccggc gataatggcc tgcttctcgc
     5221 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     5281 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     5341 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     5401 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     5461 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     5521 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5581 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     5641 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     5701 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     5761 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     5821 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     5881 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     5941 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     6001 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     6061 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     6121 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     6181 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     6241 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     6301 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     6361 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     6421 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     6481 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     6541 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     6601 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     6661 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     6721 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     6781 tgcatg
//
