LOCUS       sequence-373114-        6259 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon-optimized bacterial expression plasmid. Expresses 
            GB1-His6-TEV-CHMP4C(121-233, G154C).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6259)
  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 6259)
  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(G154C)
FEATURES             Location/Qualifiers
     source          1..6259
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     409..428
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     662..679
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    694..833
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(759..778)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     919..941
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(935..1126)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2438..3520)
                     /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     3482..3501
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3521..3598)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3804..3823
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3907..3926
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3907..3925
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3926..3950
                     /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             3965..3987
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4163..4180
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             4181..4192
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             4232..4249
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4250..4270
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(4666..4684)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4680..4727
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4902..4907,4908..4924,4925..4930))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4957..4975)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5043..5147
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5148..5216,5217..6008)
                     /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(5366..5385)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6179..6259,1..508)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
       61 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
      121 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
      181 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
      241 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
      301 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
      361 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
      421 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
      481 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
      541 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
      601 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
      661 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
      721 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
      781 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
      841 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
      901 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
      961 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     1021 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     1081 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     1141 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     1201 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     1261 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     1321 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     1381 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     1441 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     1501 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     1561 tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc
     1621 gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt
     1681 gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt
     1741 tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg
     1801 caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca
     1861 tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag
     1921 gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct
     1981 ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat
     2041 ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc
     2101 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     2161 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     2221 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     2281 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     2341 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     2401 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     2461 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     2521 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     2581 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     2641 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     2701 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     2761 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     2821 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     2881 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     2941 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     3001 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     3061 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     3121 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     3181 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     3241 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     3301 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     3361 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     3421 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     3481 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     3541 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     3601 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     3661 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     3721 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     3781 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     3841 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg
     3901 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa
     3961 taattttgtt taactttaag aaggagatat acatatgcag tataaactgg cgctgaacgg
     4021 caaaaccctg aaaggcgaaa ccaccaccga agcggtggat gcggcgaccg cggaaaaagt
     4081 gtttaaacag tatgcgaacg ataacggcgt ggatggcgaa tggacctatg atgatgcgac
     4141 caaaaccttt accgtgaccg aactggtgcc gcgcggcagc attgaaggcc gcattcgcga
     4201 aaacctgaaa gatagcggcc tgtttctgga acatcatcat catcatcatg aaaacctgta
     4261 ttttcaaggc agcgaaaaca tggatctgaa caaaattgat gatctgatgc aagaaattac
     4321 cgaacagcaa gatattgcgc aagaaattag cgaagcgttt agtcagcgcg tgtgctttgg
     4381 cgatgatttt gatgaagatg aactgatggc ggaactggaa gaactggaac aagaagaact
     4441 gaacaaaaaa atgaccaata ttcgcctgcc gaacgttccg agcagcagcc tgccggctca
     4501 gccgaaccgc aaaccgggca tgagcagcac tgcgcgtcgc tcgcgggcgg cgagcagtca
     4561 gcgcgcggaa gaagaagatg atgatattaa acagctggcg gcgtgggcga cctaaggatc
     4621 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     4681 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     4741 ctatatccgg atatcccgca agaggcccgg cagtaccggc ataaccaagc ctatgcctac
     4801 agcatccagg gtgacggtgc cgaggatgac gatgagcgca ttgttagatt tcatacacgg
     4861 tgcctgactg cgttagcaat ttaactgtga taaactaccg cattaaagct tatcgatgat
     4921 aagctgtcaa acatgagaat tcttgaagac gaaagggcct cgtgatacgc ctatttttat
     4981 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     5041 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5101 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     5161 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5221 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5281 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5341 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg
     5401 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5461 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5521 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     5581 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     5641 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     5701 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5761 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     5821 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     5881 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     5941 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6001 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     6061 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     6121 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6181 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6241 cggtggtttg tttgccgga
//
