LOCUS       sequence-161725-        5041 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Residues 205-222 of CHMP4A, Mutation disrupts binding to ALIX; 
            Bacterial Expression Vector; TEV-cleavable GST-tag; Sundquist Lab 
            Internal ID: WISP06-205.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5041)
  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 5041)
  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: GST-CHMP4A (205-222) L214A (pGEX2T(TEV))
FEATURES             Location/Qualifiers
     source          1..5041
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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             76..729
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     687..709
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             736..753
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             757..777
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(909..931)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1076..1094)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1162..1266
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1267..1335,1336..2127)
                     /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(1485..1504)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2298..2886
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2787..2806
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3040..3057
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3130..3207
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3208..4290
                     /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(3227..3246)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4339..4344,4345..4362,4363..4369)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4377..4393
                     /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     4401..4417
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4401..4417
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4413..4940
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4433..4450)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4433..4449)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4442..4464)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      121 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      181 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      241 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      301 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      361 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      421 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      481 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      541 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      601 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      661 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      721 cctccaaaat cggatctggt tccgcgtgga tctccggaaa acctgtattt ccagggccat
      781 atgcccaaag tggatgaaga tgaagaagca gcaaagcagt tggctgagtg ggtatcctga
      841 ggatcctcga gaattcatcg tgactgactg acgatctgcc tcgcgcgttt cggtgatgac
      901 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
      961 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca
     1021 gccatgaccc agtcacgtag cgatagcgga gtgtataatt cttgaagacg aaagggcctc
     1081 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     1141 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     1201 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     1261 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     1321 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     1381 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     1441 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     1501 ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     1561 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     1621 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     1681 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     1741 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     1801 acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     1861 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     1921 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     1981 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     2041 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     2101 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     2161 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     2221 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     2281 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     2341 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     2401 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     2461 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     2521 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     2581 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     2641 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     2701 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     2761 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     2821 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     2881 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     2941 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     3001 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     3061 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3121 ataaattccg acaccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg
     3181 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag
     3241 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
     3301 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
     3361 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
     3421 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
     3481 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
     3541 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
     3601 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
     3661 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
     3721 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
     3781 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
     3841 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
     3901 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
     3961 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg
     4021 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
     4081 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
     4141 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
     4201 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     4261 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac
     4321 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     4381 gagcggataa caatttcaca caggaaacag ctatgaccat gattacggat tcactggccg
     4441 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     4501 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     4561 aacagttgcg cagcctgaat ggcgaatggc gctttgcctg gtttccggca ccagaagcgg
     4621 tgccggaaag ctggctggag tgcgatcttc ctgaggccga tactgtcgtc gtcccctcaa
     4681 actggcagat gcacggttac gatgcgccca tctacaccaa cgtgacctat cccattacgg
     4741 tcaatccgcc gtttgttccc acggagaatc cgacgggttg ttactcgctc acatttaatg
     4801 ttgatgaaag ctggctacag gaaggccaga cgcgaattat ttttgatggc gttggaatta
     4861 gcttatcgac tgcacggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg
     4921 tatggctgtg caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt
     4981 ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct
     5041 g
//
