LOCUS       sequence-152693-        5650 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Residues 1-204 of CHMP4A; Bacterial Expression vector; TEV-cleavable
            GST tag; Sundquist Lab internal ID: WISP06-198.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5650)
  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 5650)
  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 (1-204) (pGEX2T(TEV))
FEATURES             Location/Qualifiers
     source          1..5650
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..935
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             939..959
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1700..1722)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1867..1885)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1953..2057
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2058..2126,2127..2918)
                     /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(2276..2295)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3089..3677
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3578..3597
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3831..3848
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3921..3998
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             3999..5081
                     /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(4018..4037)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5094..5115
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5130..5135,5136..5153,5154..5160)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5168..5184
                     /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     5192..5208
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5192..5208
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5204..5650,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5224..5241)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5224..5240)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5233..5255)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gttccgcgtg gatctccgga aaacctgtat ttccagggcc
      961 atatgagcgg tctgggccgg ctcttcggga aggggaagaa ggagaaaggg ccaacccctg
     1021 aagaagcaat acagaaactg aaggagacag agaagatact gatcaagaaa caggaatttt
     1081 tggagcagaa gattcaacag gagctacaaa cagccaagaa gtatgggacc aagaataaga
     1141 gagctgccct acaggctttg cggaggaaga aaagattcga acagcagctg gcacaaactg
     1201 acgggacatt atccaccctg gagtttcagc gtgaggccat tgagaatgcc actaccaatg
     1261 cagaagtcct tcgtaccatg gagcttgctg cccaaagcat gaagaaggcc taccaggaca
     1321 tggacattga caaggtagat gaactgatga ctgacatcac ggaacaacag gaggtggccc
     1381 agcagatctc agatgccatt tctcggccta tgggctttgg agatgatgtg gatgaggatg
     1441 aactgctgga ggagctagag gagctggagc aggaggaatt ggcccaggag ttgttaaatg
     1501 tgggcgacaa ggaagaagaa ccctcagtca aattgcctag tgtaccttct actcatctgc
     1561 cggcagggcc agcttaaaaa gtggatgaag atgaagaagc actaaagcag ttggctgagt
     1621 gggtatcctg aggatcctcg agaattcatc gtgactgact gacgatctgc ctcgcgcgtt
     1681 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     1741 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     1801 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtataat tcttgaagac
     1861 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     1921 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1981 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     2041 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     2101 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     2161 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     2221 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     2281 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     2341 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     2401 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     2461 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     2521 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     2581 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     2641 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     2701 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     2761 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     2821 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     2881 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     2941 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     3001 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     3061 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     3121 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     3181 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     3241 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     3301 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     3361 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3421 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3481 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3541 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3601 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3661 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3721 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3781 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     3841 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3901 tttcacaccg cataaattcc gacaccatcg aatggtgcaa aacctttcgc ggtatggcat
     3961 gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg
     4021 atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca
     4081 gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca
     4141 ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca
     4201 cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg
     4261 atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta
     4321 aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc
     4381 tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc
     4441 ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc
     4501 gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc
     4561 cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca
     4621 atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac
     4681 aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc
     4741 agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata
     4801 tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca
     4861 ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct
     4921 ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa
     4981 ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     5041 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     5101 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     5161 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgga
     5221 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     5281 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     5341 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc
     5401 accagaagcg gtgccggaaa gctggctgga gtgcgatctt cctgaggccg atactgtcgt
     5461 cgtcccctca aactggcaga tgcacggtta cgatgcgccc atctacacca acgtaaccta
     5521 tcccattacg gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt gttactcgct
     5581 cacatttaat gttgatgaaa gctggctaca ggaaggccag acgcgaatta tttttgatgg
     5641 cgttggaatt
//
