LOCUS       sequence-296400-        6235 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon-optimized bacterial expression plasmid. Expresses 
            GB1-His6-TEV-ALIX(703-800)-Strep..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6235)
  AUTHORS   Elias RD, Ma W, Ghirlando R, Schwieters CD, Reddy VS, Deshmukh L
  TITLE     Proline-rich domain of human ALIX contains multiple TSG101-UEV 
            interaction sites and forms phosphorylation-mediated reversible 
            amyloids.
  JOURNAL   Proc Natl Acad Sci U S A. 2020 Sep 11. pii: 2010635117. doi: 
            10.1073/pnas.2010635117.
  PUBMED    32917811
REFERENCE   2  (bases 1 to 6235)
  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: "Proc Natl 
            Acad Sci U S A. 2020 Sep 11. pii: 2010635117. doi: 
            10.1073/pnas.2010635117."
COMMENT     Sequence Label: GB1-PRD(703-800)-Strep
FEATURES             Location/Qualifiers
     source          1..6235
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             257..274
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             275..286
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             326..343
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             344..364
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             659..682
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(736..754)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      750..797
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(972..977,978..994,995..1000))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1027..1045)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1113..1217
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1218..1286,1287..2078)
                     /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(1436..1455)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2249..2837
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2738..2757
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2991..3008
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3023..3162
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3088..3107)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3248..3270
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3264..3455)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4767..5849)
                     /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     5811..5830
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5850..5927)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6133..6152
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaatacaaa ctggcgctga acggcaaaac
      121 gctgaagggt gaaacgacga ccgaggccgt tgatgcggcc acggccgaga aagtgttcaa
      181 gcagtacgcc aacgacaacg gtgttgatgg cgaatggacc tacgacgatg cgaccaaaac
      241 cttcaccgtg accgaactgg ttccgcgcgg cagtatcgaa ggtcgcatcc gcgagaatct
      301 gaaagatagc ggtctgtttc tggagcatca ccaccatcac catgaaaatc tgtacttcca
      361 aggcgacgaa ctgctgaaag atctgcagca gagcattgcc cgcgaaccaa gcgccccaag
      421 catcccgacg ccagcctatc agagtagtcc ggcgggtggt catgcgccga ccccaccaac
      481 gccggcccca cgtacgatgc cgccaaccaa accgcaacca ccagcgcgtc cgccaccacc
      541 agttctccca gccaatcgcg ccccaagtgc gaccgcgcca agcccagttg gtgcgggtac
      601 cgccgcgcca gcgccaagtc aaacgccggg tagtgcccca ccaccacaag cgcaaggctg
      661 gagccatccg cagttcgaaa agtaaggatc cggctgctaa caaagcccga aaggaagctg
      721 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
      781 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg atatcccgca agaggcccgg
      841 cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc cgaggatgac
      901 gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat ttaactgtga
      961 taaactaccg cattaaagct tatcgatgat aagctgtcaa acatgagaat tcttgaagac
     1021 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     1081 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1141 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     1201 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     1261 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     1321 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     1381 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     1441 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     1501 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     1561 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     1621 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     1681 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     1741 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     1801 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     1861 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     1921 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     1981 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     2041 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     2101 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     2161 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2221 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2281 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2341 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     2401 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     2461 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     2521 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     2581 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     2641 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     2701 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     2761 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     2821 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     2881 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     2941 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     3001 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3061 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3121 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     3181 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     3241 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     3301 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     3361 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     3421 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     3481 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     3541 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     3601 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     3661 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     3721 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     3781 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     3841 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     3901 agcacgatca tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg
     3961 ctgctggaga tggcggacgc gatggatatg ttctgccaag ggttggtttg cgcattcaca
     4021 gttctccgca agaattgatt ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc
     4081 cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga
     4141 ggcagacaag gtatagggcg gcgcctacaa tccatgccaa cccgttccat gtgctcgccg
     4201 aggcggcata aatcgccgtg acgatcagcg gtccaatgat cgaagttagg ctggtaagag
     4261 ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg
     4321 cctgcaacgc gggcatcccg atgccgccgg aagcgagaag aatcataatg gggaaggcca
     4381 tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg
     4441 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     4501 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     4561 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     4621 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     4681 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     4741 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4801 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     4861 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     4921 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     4981 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     5041 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     5101 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     5161 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     5221 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     5281 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     5341 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     5401 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     5461 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     5521 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     5581 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     5641 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     5701 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     5761 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     5821 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     5881 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     5941 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     6001 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     6061 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     6121 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     6181 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaat
//
