LOCUS       sequence-393310-        6011 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon-optimized bacterial expression plasmid. Expresses 
            GB1-His6-TEV-CHMP4C(121-233, S191C,I225A,L228A,W231A).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6011)
  AUTHORS   Elias RD, Zhu Y, Su Q, Ghirlando R, Zhang J, Deshmukh L
  TITLE     Reversible phase separation of ESCRT protein ALIX through tyrosine 
            phosphorylation.
  JOURNAL   Sci Adv. 2023 Jul 14;9(28):eadg3913. doi: 10.1126/sciadv.adg3913. 
            Epub 2023 Jul 14.
  PUBMED    37450591
REFERENCE   2  (bases 1 to 6011)
  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: "Sci Adv."; 
            date: "2023-07-14"; volume: "9(28):eadg3913. doi"; pages: " 
            10.1126/sciadv.adg3913. Epub 2023 Jul 14"
COMMENT     Sequence Label: GB1-Delta-N-CHMP4C(S191C,I225A,L228A,W231A)
FEATURES             Location/Qualifiers
     source          1..6011
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1260..2342)
                     /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     2304..2323
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2343..2420)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2626..2645
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2729..2748
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2729..2747
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2748..2772
                     /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             2787..2809
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2817..2849
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             3027..3044
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3045..3056
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3096..3113
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3114..3134
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3531..3549)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3617..3721
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3722..3790,3791..4582)
                     /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(3940..3959)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4753..5341
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5242..5261
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5495..5512
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5527..5666
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5592..5611)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5752..5774
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5768..5959)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
       61 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
      121 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
      181 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
      241 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
      301 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
      361 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggccag
      421 gacccaacgc tgcccgagat gcgccgcgtg cggctgctgg agatggcgga cgcgatggat
      481 atgttctgcc aagggttggt ttgcgcattc acagttctcc gcaagaattg attggctcca
      541 attcttggag tggtgaatcc gttagcgagg tgccgccggc ttccattcag gtcgaggtgg
      601 cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac aaggtatagg gcggcgccta
      661 caatccatgc caacccgttc catgtgctcg ccgaggcggc ataaatcgcc gtgacgatca
      721 gcggtccaat gatcgaagtt aggctggtaa gagccgcgag cgatccttga agctgtccct
      781 gatggtcgtc atctacctgc ctggacagca tggcctgcaa cgcgggcatc ccgatgccgc
      841 cggaagcgag aagaatcata atggggaagg ccatccagcc tcgcgtcgcg aacgccagca
      901 agacgtagcc cagcgcgtcg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
      961 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     1021 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     1081 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     1141 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     1201 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     1261 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     1321 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     1381 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     1441 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     1501 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     1561 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     1621 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     1681 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     1741 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     1801 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     1861 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     1921 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     1981 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     2041 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     2101 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     2161 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     2221 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     2281 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     2341 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     2401 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     2461 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     2521 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     2581 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     2641 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     2701 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
     2761 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgg
     2821 ctagcatgac tggtggacag caaatgggtc gcggatccat gcagtataaa ctggcgctga
     2881 acggcaaaac cctgaaaggc gaaaccacca ccgaagcggt ggatgcggcg accgcggaaa
     2941 aagtgtttaa acagtatgcg aacgataacg gcgtggatgg cgaatggacc tatgatgatg
     3001 cgaccaaaac ctttaccgtg accgaactgg tgccgcgcgg cagcattgaa ggccgcattc
     3061 gcgaaaacct gaaagatagc ggcctgtttc tggaacatca tcatcatcat catgaaaacc
     3121 tgtattttca aggcagcgaa aacatggatc tgaacaaaat tgatgatctg atgcaagaaa
     3181 ttaccgaaca gcaagatatt gcgcaagaaa ttagcgaagc gtttagtcag cgcgtgggct
     3241 ttggcgatga ttttgatgaa gatgaactga tggcggaact ggaagaactg gaacaagaag
     3301 aactgaacaa aaaaatgacc aatattcgcc tgccgaacgt tccgagctgc agcctgccgg
     3361 ctcagccgaa ccgcaaaccg ggcatgagca gcactgcgcg tcgctcgcgg gcggcgagca
     3421 gtcagcgcgc ggaagaagaa gatgatgatg ccaaacaggc ggcggcggcg gcgacctaaa
     3481 agcttatcga tgataagctg tcaaacatga gaattcttga agacgaaagg gcctcgtgat
     3541 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     3601 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3661 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3721 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     3781 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     3841 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     3901 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     3961 ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     4021 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     4081 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     4141 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     4201 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     4261 gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     4321 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     4381 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     4441 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4501 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4561 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     4621 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4681 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4741 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4801 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4861 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     4921 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4981 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5041 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     5101 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5161 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5221 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5281 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5341 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5401 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     5461 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     5521 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     5581 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     5641 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     5701 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     5761 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     5821 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     5881 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     5941 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     6001 tgataccgat g
//
