LOCUS       sequence-296403-        6127 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon-optimized bacterial expression plasmid. Expresses 
            GB1-His6-TEV-ALIX(800-868)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6127)
  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 6127)
  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(800-868)
FEATURES             Location/Qualifiers
     source          1..6127
                     /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"
     primer_bind     complement(628..646)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      642..689
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(864..869,870..886,887..892))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(919..937)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1005..1109
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1110..1178,1179..1970)
                     /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(1328..1347)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2141..2729
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2630..2649
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2883..2900
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2915..3054
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2980..2999)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3140..3162
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3156..3347)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4659..5741)
                     /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     5703..5722
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5742..5819)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6025..6044
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcagtacaaa ctggcgctga acggcaaaac
      121 gctgaaaggt gagaccacca ccgaagcggt tgatgcggcg accgccgaaa aggtgttcaa
      181 acagtacgcc aacgacaacg gtgtggatgg cgaatggacc tacgatgacg ccaccaaaac
      241 gttcaccgtg accgaactgg ttccgcgcgg tagcattgaa ggccgcatcc gcgaaaatct
      301 gaaagatagc ggtctgtttc tggaacatca tcaccaccat cacgaaaatc tgtacttcca
      361 aggcggcggt ccgccgtatc caacctaccc gggttacccg ggctactgcc agatgccgat
      421 gccaatgggc tacaacccgt acgcctacgg ccagtacaat atgccgtacc cgccggtgta
      481 ccatcagagt ccgggccaag ccccatatcc gggtccacag cagccaagtt acccgtttcc
      541 gcagccgccg cagcagagtt actatccgca gcagtaagga tccggctgct aacaaagccc
      601 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
      661 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggatatcccg
      721 caagaggccc ggcagtaccg gcataaccaa gcctatgcct acagcatcca gggtgacggt
      781 gccgaggatg acgatgagcg cattgttaga tttcatacac ggtgcctgac tgcgttagca
      841 atttaactgt gataaactac cgcattaaag cttatcgatg ataagctgtc aaacatgaga
      901 attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
      961 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1021 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     1081 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     1141 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     1201 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     1261 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     1321 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc
     1381 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     1441 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     1501 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     1561 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     1621 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
     1681 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     1741 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     1801 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     1861 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     1921 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     1981 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     2041 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     2101 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2161 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2221 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2281 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     2341 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     2401 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     2461 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     2521 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     2581 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     2641 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     2701 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     2761 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     2821 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     2881 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     2941 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     3001 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     3061 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3121 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3181 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     3241 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     3301 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     3361 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     3421 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     3481 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     3541 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     3601 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     3661 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     3721 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     3781 ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgagatg
     3841 cgccgcgtgc ggctgctgga gatggcggac gcgatggata tgttctgcca agggttggtt
     3901 tgcgcattca cagttctccg caagaattga ttggctccaa ttcttggagt ggtgaatccg
     3961 ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac
     4021 gcaacgcggg gaggcagaca aggtataggg cggcgcctac aatccatgcc aacccgttcc
     4081 atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccaatg atcgaagtta
     4141 ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca tctacctgcc
     4201 tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa
     4261 tggggaaggc catccagcct cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg
     4321 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     4381 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     4441 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     4501 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     4561 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     4621 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4681 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4741 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     4801 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     4861 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     4921 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     4981 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     5041 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     5101 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     5161 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     5221 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     5281 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     5341 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     5401 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     5461 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     5521 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     5581 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     5641 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     5701 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     5761 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     5821 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     5881 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     5941 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     6001 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     6061 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
     6121 gcgaaat
//
