LOCUS       sequence-373089-        6259 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon-optimized bacterial expression plasmid. Expresses 
            GB1-His6-TEV-CHMP4C(121-233, M165C).
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(M165C)
FEATURES             Location/Qualifiers
     source          1..6259
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     189..208
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     442..459
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    474..613
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(539..558)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     699..721
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(715..906)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2218..3300)
                     /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     3262..3281
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3301..3378)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3584..3603
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3687..3706
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3687..3705
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3706..3730
                     /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             3745..3767
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3943..3960
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3961..3972
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             4012..4029
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4030..4050
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(4446..4464)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4460..4507
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4682..4687,4688..4704,4705..4710))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4737..4755)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4823..4927
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4928..4996,4997..5788)
                     /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(5146..5165)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5959..6259,1..288)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
       61 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
      121 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
      181 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
      241 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
      301 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
      361 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
      421 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
      481 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
      541 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
      601 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
      661 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
      721 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
      781 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
      841 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
      901 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
      961 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     1021 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     1081 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     1141 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     1201 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     1261 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     1321 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggccagga
     1381 cccaacgctg cccgagatgc gccgcgtgcg gctgctggag atggcggacg cgatggatat
     1441 gttctgccaa gggttggttt gcgcattcac agttctccgc aagaattgat tggctccaat
     1501 tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc
     1561 cggctccatg caccgcgacg caacgcgggg aggcagacaa ggtatagggc ggcgcctaca
     1621 atccatgcca acccgttcca tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc
     1681 ggtccaatga tcgaagttag gctggtaaga gccgcgagcg atccttgaag ctgtccctga
     1741 tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg cgggcatccc gatgccgccg
     1801 gaagcgagaa gaatcataat ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag
     1861 acgtagccca gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     1921 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     1981 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     2041 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     2101 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     2161 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     2221 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     2281 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     2341 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     2401 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     2461 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     2521 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     2581 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     2641 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     2701 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     2761 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     2821 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     2881 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     2941 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     3001 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     3061 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     3121 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     3181 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     3241 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     3301 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     3361 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     3421 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     3481 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     3541 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     3601 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     3661 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     3721 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat acatatgcag
     3781 tataaactgg cgctgaacgg caaaaccctg aaaggcgaaa ccaccaccga agcggtggat
     3841 gcggcgaccg cggaaaaagt gtttaaacag tatgcgaacg ataacggcgt ggatggcgaa
     3901 tggacctatg atgatgcgac caaaaccttt accgtgaccg aactggtgcc gcgcggcagc
     3961 attgaaggcc gcattcgcga aaacctgaaa gatagcggcc tgtttctgga acatcatcat
     4021 catcatcatg aaaacctgta ttttcaaggc agcgaaaaca tggatctgaa caaaattgat
     4081 gatctgatgc aagaaattac cgaacagcaa gatattgcgc aagaaattag cgaagcgttt
     4141 agtcagcgcg tgggctttgg cgatgatttt gatgaagatg aactgtgcgc ggaactggaa
     4201 gaactggaac aagaagaact gaacaaaaaa atgaccaata ttcgcctgcc gaacgttccg
     4261 agcagcagcc tgccggctca gccgaaccgc aaaccgggca tgagcagcac tgcgcgtcgc
     4321 tcgcgggcgg cgagcagtca gcgcgcggaa gaagaagatg atgatattaa acagctggcg
     4381 gcgtgggcga cctaaggatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     4441 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     4501 ttttttgctg aaaggaggaa ctatatccgg atatcccgca agaggcccgg cagtaccggc
     4561 ataaccaagc ctatgcctac agcatccagg gtgacggtgc cgaggatgac gatgagcgca
     4621 ttgttagatt tcatacacgg tgcctgactg cgttagcaat ttaactgtga taaactaccg
     4681 cattaaagct tatcgatgat aagctgtcaa acatgagaat tcttgaagac gaaagggcct
     4741 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     4801 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4861 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4921 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     4981 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5041 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5101 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     5161 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     5221 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     5281 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     5341 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     5401 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     5461 cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     5521 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     5581 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     5641 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     5701 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     5761 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     5821 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     5881 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5941 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6001 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6061 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     6121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     6241 acgatagtta ccggataag
//
