LOCUS       sequence-371391-        5843 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon-optimized bacterial expression plasmid. Expresses 
            GB1-His6-TEV-CEP55(160-217, S215C).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5843)
  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 5843)
  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-CEP55(160-217, S215C)
FEATURES             Location/Qualifiers
     source          1..5843
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      30..618
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     519..538
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     772..789
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    804..943
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(869..888)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1029..1051
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1045..1236)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2548..3630)
                     /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     3592..3611
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3631..3708)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3914..3933
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4017..4036
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4017..4035
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4036..4060
                     /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             4075..4097
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4105..4137
                     /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             4315..4332
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             4333..4344
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             4384..4401
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4402..4422
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(4651..4669)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4737..4841
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4842..4910,4911..5702)
                     /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(5060..5079)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
       61 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
      121 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
      181 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
      241 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
      301 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
      361 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
      421 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
      481 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
      541 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
      601 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
      661 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
      721 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
      781 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
      841 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
      901 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
      961 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     1021 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     1081 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     1141 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     1201 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     1261 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     1321 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     1381 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     1441 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     1501 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     1561 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     1621 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     1681 gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc gccgcgtgcg
     1741 gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt gcgcattcac
     1801 agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt tagcgaggtg
     1861 ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg
     1921 aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca tgtgctcgcc
     1981 gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag gctggtaaga
     2041 gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct ggacagcatg
     2101 gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat ggggaaggcc
     2161 atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc cgccatgccg
     2221 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     2281 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     2341 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     2401 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     2461 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     2521 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     2581 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     2641 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     2701 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     2761 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     2821 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     2881 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     2941 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     3001 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     3061 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     3121 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     3181 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     3241 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     3301 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     3361 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     3421 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     3481 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     3541 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     3601 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     3661 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     3721 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     3781 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     3841 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     3901 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     3961 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat
     4021 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
     4081 taactttaag aaggagatat acatatggct agcatgactg gtggacagca aatgggtcgc
     4141 ggatccatgc agtataaact ggcgctgaac ggcaaaaccc tgaaaggcga aaccaccacc
     4201 gaagcggtgg atgcggcgac cgcggaaaaa gtgtttaaac agtatgcgaa cgataacggc
     4261 gtggatggcg aatggaccta tgatgatgcg accaaaacct ttaccgtgac cgaactggtg
     4321 ccgcgcggca gcattgaagg ccgcattcgc gaaaacctga aagatagcgg cctgtttctg
     4381 gaacatcatc atcatcatca tgaaaacctg tattttcaag gcagctttaa cagcagcatt
     4441 aacaacattc atgaaatgga aattcagctg aaagatgcgc tggaaaaaaa tcagcagtgg
     4501 ctggtgtatg atcagcagcg cgaagtgtat gtgaaaggcc tgctggcgaa aatttttgaa
     4561 ctggaaaaaa aaaccgaaac cgcggcgcat tgcctgtaaa agcttatcga tgataagctg
     4621 tcaaacatga gaattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta
     4681 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     4741 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     4801 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     4861 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     4921 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     4981 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     5041 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
     5101 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5161 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     5221 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5281 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     5341 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     5401 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     5461 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     5521 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     5581 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     5641 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     5701 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     5761 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     5821 agttttcgtt ccactgagcg tca
//
