LOCUS       sequence-367897-        8269 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon-optimized bacterial expression plasmid. Expresses ALIX(1-868, 
            P801G)-Strep. P801G mutation facilitates expression in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8269)
  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 8269)
  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: ALIX(P801G)-Strep
FEATURES             Location/Qualifiers
     source          1..8269
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     79..98
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     332..349
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    364..503
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(429..448)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     589..611
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(605..796)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2108..3190)
                     /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     3152..3171
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3191..3268)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3474..3493
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3577..3596
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3577..3595
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3596..3620
                     /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             3635..3657
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6269..6292
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(6346..6364)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6360..6407
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6582..6587,6588..6604,6605..6610))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6637..6655)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6723..6827
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6828..6896,6897..7688)
                     /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(7046..7065)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7859..8269,1..178)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
       61 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
      121 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
      181 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
      241 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
      301 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      361 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
      421 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
      481 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
      541 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
      601 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
      661 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
      721 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
      781 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
      841 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
      901 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
      961 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     1021 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     1081 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     1141 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     1201 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     1261 gtggccagga cccaacgctg cccgagatgc gccgcgtgcg gctgctggag atggcggacg
     1321 cgatggatat gttctgccaa gggttggttt gcgcattcac agttctccgc aagaattgat
     1381 tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt
     1441 cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa ggtatagggc
     1501 ggcgcctaca atccatgcca acccgttcca tgtgctcgcc gaggcggcat aaatcgccgt
     1561 gacgatcagc ggtccaatga tcgaagttag gctggtaaga gccgcgagcg atccttgaag
     1621 ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg cgggcatccc
     1681 gatgccgccg gaagcgagaa gaatcataat ggggaaggcc atccagcctc gcgtcgcgaa
     1741 cgccagcaag acgtagccca gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc
     1801 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     1861 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     1921 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     1981 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     2041 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     2101 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     2161 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     2221 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     2281 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     2341 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     2401 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     2461 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     2521 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     2581 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     2641 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     2701 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     2761 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     2821 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     2881 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     2941 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     3001 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     3061 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     3121 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     3181 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     3241 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     3301 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     3361 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     3421 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     3481 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     3541 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
     3601 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
     3661 acatatggcg acctttatta gcgtgcagct gaaaaaaacg agcgaagtgg atctggcgaa
     3721 accgctggtg aaatttattc agcagaccta tccgagcggc ggcgaagaac aagcgcagta
     3781 ttgtcgcgca gctgaagagc tgagcaaatt gcgccgcgcc gcggttggtc gcccgctgga
     3841 taaacatgaa ggcgcgctgg aaaccctgct gcgctattat gatcagattt gcagcattga
     3901 accgaaattt ccgtttagcg aaaatcagat ttgcctgacc tttacctgga aagatgcgtt
     3961 tgataaaggc agcctgtttg gcggcagcgt gaaactggct ctggctagcc tgggctatga
     4021 aaaaagctgc gtgctgttta actgcgcggc cctggcgagt cagattgcgg cggaacagaa
     4081 cctggataac gatgaaggcc tgaaaattgc agcgaagcat taccaatttg cgagcggcgc
     4141 gtttctgcat attaaagaaa ccgtgctgag cgcgctgagc cgcgaaccga ccgtggatat
     4201 tagcccggat accgtgggca ccctgagcct gattatgctg gcgcaagcac aagaagtgtt
     4261 ttttctgaaa gcgacccgcg ataaaatgaa agatgcgatt attgcgaaac tggcgaacca
     4321 agcggcggat tattttggcg atgcgtttaa acagtgtcag tataaagata ccctgccgaa
     4381 agaagtgttt ccggtgctgg cggcgaaaca ttgtataatg caagcgaacg cggaatatca
     4441 ccaaagtatc ttggcgaagc agcagaaaaa atttggcgaa gaaattgcgc gcctgcagca
     4501 tgcggcggaa ctgattaaaa ccgtggcgag ccgctatgat gaatatgtga acgtgaaaga
     4561 ttttagcgat aaaattaacc gcgcgctggc ggcggcgaaa aaagataacg attttattta
     4621 tcatgatcgc gtgccggatc tgaaagacct ggacccgatt ggcaaagcga ccctggtgaa
     4681 aagcaccccg gtgaacgtgc cgattagtca gaaatttacc gatctgtttg aaaaaatggt
     4741 gccggtgagc gtgcagcaga gcctggcggc gtataatcag cgcaaagcgg atctggtgaa
     4801 ccgcagcatt gcgcagatgc gcgaagcgac caccctggcg aacggcgtgc tggcgagcct
     4861 gaacctgccg gcggcgattg aagatgtgag cggcgatacc gtgccgcaat ctatcctgac
     4921 caaaagccgc agcgtgattg aacaaggcgg cattcagacc gtggatcagc tgattaaaga
     4981 actgccggaa ctgctgcaac gcaatcgcga aattctggat gaaagcctgc gcctgctgga
     5041 tgaagaagaa gcgaccgata acgatctgcg cgcgaaattt aaagaacgct ggcagcgcac
     5101 cccgagcaac gaactgtata aaccgctgcg cgcggaaggc accaactttc gcaccgtgct
     5161 ggataaagcg gtgcaagcgg atggccaagt gaaagaatgc tatcagagcc atcgcgatac
     5221 cattgtgctg ctgtgcaaac cggaaccgga actgaacgcg gcgattccga gcgcgaaccc
     5281 ggcgaaaacc atgcaaggca gcgaagtggt gaacgtgctg aaaagcctgc tgagcaacct
     5341 ggatgaagtg aaaaaagaac gcgaaggcct ggaaaacgat ctgaaaagcg tgaactttga
     5401 tatgacgagc aaatttctga ccgcgctggc gcaagatggc gtgattaacg aagaagcgct
     5461 gagcgtgacc gaactggatc gcgtgtatgg cggcctgacc accaaagtgc aagaaagcct
     5521 gaaaaaacaa gaaggcctgc tgaaaaacat tcaagtgagc catcaagaat ttagcaaaat
     5581 gaaacagagc aacaacgaag cgaacctgcg cgaagaagtg ctgaaaaacc tggcgaccgc
     5641 gtatgataac tttgtggaac tggtggcgaa cctgaaagaa ggcaccaaat tttataacga
     5701 actgaccgaa attctggtgc gctttcagaa caaatgcagc gatattgtgt ttgcgcgcaa
     5761 aaccgaacgc gacgaactgc tgaaagatct gcagcagagc attgcccgcg aaccaagcgc
     5821 cccaagcatc ccgacgccag cctatcagag tagtccggcg ggtggtcatg cgccgacccc
     5881 accaacgccg gccccacgta cgatgccgcc aaccaaaccg caaccaccag cgcgtccgcc
     5941 accaccagtt ctcccagcca atcgcgcccc aagtgcgacc gcgccaagcc cagttggtgc
     6001 gggtaccgcc gcgccagcgc caagtcaaac gccgggtagt gccccaccac cacaagcgca
     6061 aggcggccca tatccgacgt acccgggcta tccgggctac tgccagatgc caatgccgat
     6121 gggttacaac ccatacgcgt acggccagta taacatgccg tacccaccgg tgtaccacca
     6181 gagtccgggc caagccccat acccgggtcc acagcagcca agctacccgt tcccgcagcc
     6241 accacagcaa agttactacc cgcagcaatg gagccatccg cagttcgaaa agtaaggatc
     6301 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     6361 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     6421 ctatatccgg atatcccgca agaggcccgg cagtaccggc ataaccaagc ctatgcctac
     6481 agcatccagg gtgacggtgc cgaggatgac gatgagcgca ttgttagatt tcatacacgg
     6541 tgcctgactg cgttagcaat ttaactgtga taaactaccg cattaaagct tatcgatgat
     6601 aagctgtcaa acatgagaat tcttgaagac gaaagggcct cgtgatacgc ctatttttat
     6661 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     6721 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     6781 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     6841 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     6901 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     6961 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     7021 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg
     7081 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     7141 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     7201 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     7261 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     7321 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     7381 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     7441 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     7501 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     7561 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     7621 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     7681 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     7741 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     7801 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     7861 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     7921 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     7981 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     8041 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     8101 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     8161 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     8221 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgct
//
