LOCUS       sequence-324033-        6439 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon-optimized bacterial expression plasmid. Expresses 
            GB1-His6-TEV-ALIX(703-868, A756C, P801G, C813S)-Strep..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6439)
  AUTHORS   Elias RD, Ramaraju B, Deshmukh L
  TITLE     Mechanistic Roles of Tyrosine Phosphorylation in Reversible 
            Amyloids, Autoinhibition, and Endosomal Membrane Association of 
            ALIX.
  JOURNAL   J Biol Chem. 2021 Oct 21:101328. doi: 10.1016/j.jbc.2021.101328.
  PUBMED    34688656
REFERENCE   2  (bases 1 to 6439)
  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: "J Biol 
            Chem. 2021 Oct 21:101328. doi: 10.1016/j.jbc.2021.101328."
COMMENT     Sequence Label: GB1-PRD(703-868, A756C, P801G, C813S)-Strep
FEATURES             Location/Qualifiers
     source          1..6439
                     /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             863..886
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(940..958)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      954..1001
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1176..1181,1182..1198,1199..1204))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1231..1249)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1317..1421
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1422..1490,1491..2282)
                     /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(1640..1659)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2453..3041
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2942..2961
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3195..3212
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3227..3366
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3292..3311)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3452..3474
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3468..3659)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4971..6053)
                     /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     6015..6034
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6054..6131)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6337..6356
                     /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 ccatgtcgtc cgccaccacc
      541 agttctccca gccaatcgcg ccccaagtgc gaccgcgcca agcccagttg gtgcgggtac
      601 cgccgcgcca gcgccaagtc aaacgccggg tagtgcccca ccaccacaag cgcaaggcgg
      661 cccatatccg acgtacccgg gctatccggg ctacagtcag atgccaatgc cgatgggtta
      721 caacccatac gcgtacggcc agtataacat gccgtaccca ccggtgtacc accagagtcc
      781 gggccaagcc ccatacccgg gtccacagca gccaagctac ccgttcccgc agccaccaca
      841 gcaaagttac tacccgcagc aatggagcca tccgcagttc gaaaagtaag gatccggctg
      901 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
      961 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     1021 ccggatatcc cgcaagaggc ccggcagtac cggcataacc aagcctatgc ctacagcatc
     1081 cagggtgacg gtgccgagga tgacgatgag cgcattgtta gatttcatac acggtgcctg
     1141 actgcgttag caatttaact gtgataaact accgcattaa agcttatcga tgataagctg
     1201 tcaaacatga gaattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta
     1261 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     1321 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     1381 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     1441 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     1501 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     1561 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     1621 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
     1681 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     1741 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     1801 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     1861 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     1921 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     1981 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     2041 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2101 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     2161 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     2221 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     2281 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     2341 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     2401 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2461 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2521 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2581 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2641 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     2701 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     2761 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     2821 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     2881 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     2941 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3001 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3061 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3121 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3181 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     3241 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     3301 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     3361 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3421 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3481 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     3541 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     3601 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     3661 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     3721 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     3781 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     3841 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     3901 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     3961 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     4021 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     4081 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg
     4141 ctgcccgaga tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc
     4201 caagggttgg tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga
     4261 gtggtgaatc cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc
     4321 atgcaccgcg acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg
     4381 ccaacccgtt ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccaa
     4441 tgatcgaagt taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt
     4501 catctacctg cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga
     4561 gaagaatcat aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc
     4621 ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     4681 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     4741 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     4801 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     4861 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     4921 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     4981 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5041 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     5101 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     5161 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     5221 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
     5281 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     5341 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     5401 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     5461 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     5521 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     5581 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     5641 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     5701 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     5761 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     5821 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     5881 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     5941 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     6001 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     6061 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     6121 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     6181 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
     6241 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
     6301 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
     6361 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
     6421 gatctcgatc ccgcgaaat
//
