LOCUS       sequence-367901-        6415 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon-optimized bacterial expression plasmid. Expresses 
            GB1-His6-TEV-ALIX(703-868, P801G). P801G mutation facilitates 
            expression in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6415)
  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 6415)
  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-PRD(703-868, P801G)
FEATURES             Location/Qualifiers
     source          1..6415
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(97..116)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      910..1498
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1399..1418
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1652..1669
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1684..1823
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1749..1768)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1909..1931
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1925..2116)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3428..4510)
                     /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     4472..4491
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4511..4588)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4794..4813
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4897..4916
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4897..4915
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4916..4940
                     /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             4955..4977
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5153..5170
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5171..5182
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             5222..5239
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5240..5260
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(5812..5830)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5826..5873
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6048..6053,6054..6070,6071..6076))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6103..6121)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6189..6293
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6294..6362,6363..6415,1..739)
                     /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"
ORIGIN
        1 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
       61 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
      121 tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
      181 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
      241 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
      301 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
      361 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
      421 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
      481 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
      541 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
      601 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
      661 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
      721 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
      781 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
      841 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
      901 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
      961 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1021 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     1081 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1141 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1201 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     1261 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     1321 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     1381 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     1441 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     1501 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     1561 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     1621 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     1681 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     1741 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     1801 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     1861 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     1921 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     1981 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     2041 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     2101 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     2161 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     2221 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     2281 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     2341 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     2401 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     2461 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     2521 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     2581 gtggccagga cccaacgctg cccgagatgc gccgcgtgcg gctgctggag atggcggacg
     2641 cgatggatat gttctgccaa gggttggttt gcgcattcac agttctccgc aagaattgat
     2701 tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt
     2761 cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa ggtatagggc
     2821 ggcgcctaca atccatgcca acccgttcca tgtgctcgcc gaggcggcat aaatcgccgt
     2881 gacgatcagc ggtccaatga tcgaagttag gctggtaaga gccgcgagcg atccttgaag
     2941 ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg cgggcatccc
     3001 gatgccgccg gaagcgagaa gaatcataat ggggaaggcc atccagcctc gcgtcgcgaa
     3061 cgccagcaag acgtagccca gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc
     3121 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     3181 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     3241 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     3301 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     3361 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     3421 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     3481 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     3541 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     3601 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     3661 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     3721 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     3781 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     3841 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     3901 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     3961 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     4021 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     4081 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     4141 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     4201 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     4261 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     4321 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     4381 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     4441 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     4501 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     4561 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     4621 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     4681 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     4741 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     4801 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     4861 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
     4921 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
     4981 acatatgcaa tacaaactgg cgctgaacgg caaaacgctg aagggtgaaa cgacgaccga
     5041 ggccgttgat gcggccacgg ccgagaaagt gttcaagcag tacgccaacg acaacggtgt
     5101 tgatggcgaa tggacctacg acgatgcgac caaaaccttc accgtgaccg aactggttcc
     5161 gcgcggcagt atcgaaggtc gcatccgcga gaatctgaaa gatagcggtc tgtttctgga
     5221 gcatcaccac catcaccatg aaaatctgta cttccaaggc gacgaactgc tgaaagatct
     5281 gcagcagagc attgcccgcg aaccaagcgc cccaagcatc ccgacgccag cctatcagag
     5341 tagtccggcg ggtggtcatg cgccgacccc accaacgccg gccccacgta cgatgccgcc
     5401 aaccaaaccg caaccaccag cgcgtccgcc accaccagtt ctcccagcca atcgcgcccc
     5461 aagtgcgacc gcgccaagcc cagttggtgc gggtaccgcc gcgccagcgc caagtcaaac
     5521 gccgggtagt gccccaccac cacaagcgca aggcggccca tatccgacgt acccgggcta
     5581 tccgggctac tgccagatgc caatgccgat gggttacaac ccatacgcgt acggccagta
     5641 taacatgccg tacccaccgg tgtaccacca gagtccgggc caagccccat acccgggtcc
     5701 acagcagcca agctacccgt tcccgcagcc accacagcaa agttactacc cgcagcaata
     5761 aggatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     5821 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     5881 gaggaactat atccggatat cccgcaagag gcccggcagt accggcataa ccaagcctat
     5941 gcctacagca tccagggtga cggtgccgag gatgacgatg agcgcattgt tagatttcat
     6001 acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt aaagcttatc
     6061 gatgataagc tgtcaaacat gagaattctt gaagacgaaa gggcctcgtg atacgcctat
     6121 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     6181 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     6241 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     6301 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     6361 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacg
//
