LOCUS       sequence-367899-        6011 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon-optimized bacterial expression plasmid. Expresses 
            GB1-His6-TEV-CHMP4C(121-233, S191C)..
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)
FEATURES             Location/Qualifiers
     source          1..6011
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1451..2533)
                     /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     2495..2514
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2534..2611)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2817..2836
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2920..2939
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2920..2938
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2939..2963
                     /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             2978..3000
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3008..3040
                     /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             3218..3235
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3236..3247
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3287..3304
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3305..3325
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3722..3740)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3808..3912
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3913..3981,3982..4773)
                     /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(4131..4150)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4944..5532
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5433..5452
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5686..5703
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5718..5857
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5783..5802)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5943..5965
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(5959..6011,1..139))
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
       61 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
      121 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
      181 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
      241 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
      301 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
      361 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
      421 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
      481 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
      541 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
      601 cccgtggcca ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg gagatggcgg
      661 acgcgatgga tatgttctgc caagggttgg tttgcgcatt cacagttctc cgcaagaatt
      721 gattggctcc aattcttgga gtggtgaatc cgttagcgag gtgccgccgg cttccattca
      781 ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga caaggtatag
      841 ggcggcgcct acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg cataaatcgc
      901 cgtgacgatc agcggtccaa tgatcgaagt taggctggta agagccgcga gcgatccttg
      961 aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgca acgcgggcat
     1021 cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc ctcgcgtcgc
     1081 gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt
     1141 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     1201 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     1261 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     1321 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     1381 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     1441 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     1501 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     1561 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     1621 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     1681 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     1741 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     1801 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     1861 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     1921 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     1981 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     2041 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     2101 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     2161 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     2221 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     2281 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     2341 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     2401 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     2461 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     2521 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     2581 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     2641 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     2701 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     2761 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     2821 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     2881 cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg
     2941 aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga
     3001 tatacatatg gctagcatga ctggtggaca gcaaatgggt cgcggatcca tgcagtataa
     3061 actggcgctg aacggcaaaa ccctgaaagg cgaaaccacc accgaagcgg tggatgcggc
     3121 gaccgcggaa aaagtgttta aacagtatgc gaacgataac ggcgtggatg gcgaatggac
     3181 ctatgatgat gcgaccaaaa cctttaccgt gaccgaactg gtgccgcgcg gcagcattga
     3241 aggccgcatt cgcgaaaacc tgaaagatag cggcctgttt ctggaacatc atcatcatca
     3301 tcatgaaaac ctgtattttc aaggcagcga aaacatggat ctgaacaaaa ttgatgatct
     3361 gatgcaagaa attaccgaac agcaagatat tgcgcaagaa attagcgaag cgtttagtca
     3421 gcgcgtgggc tttggcgatg attttgatga agatgaactg atggcggaac tggaagaact
     3481 ggaacaagaa gaactgaaca aaaaaatgac caatattcgc ctgccgaacg ttccgagctg
     3541 cagcctgccg gctcagccga accgcaaacc gggcatgagc agcactgcgc gtcgctcgcg
     3601 ggcggcgagc agtcagcgcg cggaagaaga agatgatgat attaaacagc tggcggcgtg
     3661 ggcgacctaa aagcttatcg atgataagct gtcaaacatg agaattcttg aagacgaaag
     3721 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     3781 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3841 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     3901 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     3961 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     4021 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     4081 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     4141 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     4201 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     4261 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     4321 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     4381 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     4441 gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     4501 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     4561 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     4621 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     4681 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     4741 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     4801 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     4861 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     4921 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4981 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     5041 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     5101 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     5161 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     5221 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     5281 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     5341 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     5401 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     5461 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     5521 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     5581 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     5641 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     5701 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     5761 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     5821 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     5881 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     5941 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     6001 gcggtaaagc t
//
