LOCUS       sequence-374494-        8263 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon-optimized bacterial expression plasmid. Expresses 
            ALIX-His(1-868, P801G)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8263)
  AUTHORS   Nelson SL, Li Y, Chen Y, Deshmukh L
  TITLE     Avidity-Based Method for the Efficient Generation of 
            Monoubiquitinated Recombinant Proteins.
  JOURNAL   J Am Chem Soc. 2023 Apr 3. doi: 10.1021/jacs.3c01943.
  PUBMED    37010382
REFERENCE   2  (bases 1 to 8263)
  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 Am Chem 
            Soc. 2023 Apr 3. doi: 10.1021/jacs.3c01943."
COMMENT     Sequence Label: ALIX-His(1-868, P801G)
FEATURES             Location/Qualifiers
     source          1..8263
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      442..1030
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     931..950
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1184..1201
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1216..1355
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1281..1300)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1441..1463
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1457..1648)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2960..4042)
                     /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     4004..4023
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4043..4120)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4326..4345
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4429..4448
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4429..4447
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4448..4472
                     /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             4487..4509
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7121..7138
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7192..7210)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7206..7253
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(7428..7433,7434..7450,7451..7456))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(7483..7501)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7569..7673
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7674..7742,7743..8263,1..271)
                     /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(7892..7911)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
       61 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
      121 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
      181 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
      241 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
      301 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
      361 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
      421 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
      481 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
      541 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
      601 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
      661 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
      721 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
      781 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
      841 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
      901 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
      961 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     1021 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     1081 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     1141 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     1201 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     1261 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     1321 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     1381 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     1441 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     1501 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     1561 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     1621 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     1681 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     1741 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     1801 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     1861 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     1921 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     1981 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     2041 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     2101 tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg cggctgctgg
     2161 agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc acagttctcc
     2221 gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg tgccgccggc
     2281 ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac
     2341 aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg ccgaggcggc
     2401 ataaatcgcc gtgacgatca gcggtccaat gatcgaagtt aggctggtaa gagccgcgag
     2461 cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca tggcctgcaa
     2521 cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg ccatccagcc
     2581 tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc cggcgataat
     2641 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     2701 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     2761 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     2821 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     2881 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     2941 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3001 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     3061 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     3121 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     3181 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     3241 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     3301 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     3361 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     3421 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     3481 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     3541 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     3601 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     3661 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     3721 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     3781 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     3841 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     3901 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     3961 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     4021 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     4081 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     4141 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     4201 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     4261 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     4321 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     4381 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca
     4441 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta
     4501 agaaggagat atacatatgg cgacctttat tagcgtgcag ctgaaaaaaa cgagcgaagt
     4561 ggatctggcg aaaccgctgg tgaaatttat tcagcagacc tatccgagcg gcggcgaaga
     4621 acaagcgcag tattgtcgcg cagctgaaga gctgagcaaa ttgcgccgcg ccgcggttgg
     4681 tcgcccgctg gataaacatg aaggcgcgct ggaaaccctg ctgcgctatt atgatcagat
     4741 ttgcagcatt gaaccgaaat ttccgtttag cgaaaatcag atttgcctga cctttacctg
     4801 gaaagatgcg tttgataaag gcagcctgtt tggcggcagc gtgaaactgg ctctggctag
     4861 cctgggctat gaaaaaagct gcgtgctgtt taactgcgcg gccctggcga gtcagattgc
     4921 ggcggaacag aacctggata acgatgaagg cctgaaaatt gcagcgaagc attaccaatt
     4981 tgcgagcggc gcgtttctgc atattaaaga aaccgtgctg agcgcgctga gccgcgaacc
     5041 gaccgtggat attagcccgg ataccgtggg caccctgagc ctgattatgc tggcgcaagc
     5101 acaagaagtg ttttttctga aagcgacccg cgataaaatg aaagatgcga ttattgcgaa
     5161 actggcgaac caagcggcgg attattttgg cgatgcgttt aaacagtgtc agtataaaga
     5221 taccctgccg aaagaagtgt ttccggtgct ggcggcgaaa cattgtataa tgcaagcgaa
     5281 cgcggaatat caccaaagta tcttggcgaa gcagcagaaa aaatttggcg aagaaattgc
     5341 gcgcctgcag catgcggcgg aactgattaa aaccgtggcg agccgctatg atgaatatgt
     5401 gaacgtgaaa gattttagcg ataaaattaa ccgcgcgctg gcggcggcga aaaaagataa
     5461 cgattttatt tatcatgatc gcgtgccgga tctgaaagac ctggacccga ttggcaaagc
     5521 gaccctggtg aaaagcaccc cggtgaacgt gccgattagt cagaaattta ccgatctgtt
     5581 tgaaaaaatg gtgccggtga gcgtgcagca gagcctggcg gcgtataatc agcgcaaagc
     5641 ggatctggtg aaccgcagca ttgcgcagat gcgcgaagcg accaccctgg cgaacggcgt
     5701 gctggcgagc ctgaacctgc cggcggcgat tgaagatgtg agcggcgata ccgtgccgca
     5761 atctatcctg accaaaagcc gcagcgtgat tgaacaaggc ggcattcaga ccgtggatca
     5821 gctgattaaa gaactgccgg aactgctgca acgcaatcgc gaaattctgg atgaaagcct
     5881 gcgcctgctg gatgaagaag aagcgaccga taacgatctg cgcgcgaaat ttaaagaacg
     5941 ctggcagcgc accccgagca acgaactgta taaaccgctg cgcgcggaag gcaccaactt
     6001 tcgcaccgtg ctggataaag cggtgcaagc ggatggccaa gtgaaagaat gctatcagag
     6061 ccatcgcgat accattgtgc tgctgtgcaa accggaaccg gaactgaacg cggcgattcc
     6121 gagcgcgaac ccggcgaaaa ccatgcaagg cagcgaagtg gtgaacgtgc tgaaaagcct
     6181 gctgagcaac ctggatgaag tgaaaaaaga acgcgaaggc ctggaaaacg atctgaaaag
     6241 cgtgaacttt gatatgacga gcaaatttct gaccgcgctg gcgcaagatg gcgtgattaa
     6301 cgaagaagcg ctgagcgtga ccgaactgga tcgcgtgtat ggcggcctga ccaccaaagt
     6361 gcaagaaagc ctgaaaaaac aagaaggcct gctgaaaaac attcaagtga gccatcaaga
     6421 atttagcaaa atgaaacaga gcaacaacga agcgaacctg cgcgaagaag tgctgaaaaa
     6481 cctggcgacc gcgtatgata actttgtgga actggtggcg aacctgaaag aaggcaccaa
     6541 attttataac gaactgaccg aaattctggt gcgctttcag aacaaatgca gcgatattgt
     6601 gtttgcgcgc aaaaccgaac gcgacgaact gctgaaagat ctgcagcaga gcattgcccg
     6661 cgaaccaagc gccccaagca tcccgacgcc agcctatcag agtagtccgg cgggtggtca
     6721 tgcgccgacc ccaccaacgc cggccccacg tacgatgccg ccaaccaaac cgcaaccacc
     6781 agcgcgtccg ccaccaccag ttctcccagc caatcgcgcc ccaagtgcga ccgcgccaag
     6841 cccagttggt gcgggtaccg ccgcgccagc gccaagtcaa acgccgggta gtgccccacc
     6901 accacaagcg caaggcggcc catatccgac gtacccgggc tatccgggct actgccagat
     6961 gccaatgccg atgggttaca acccatacgc gtacggccag tataacatgc cgtacccacc
     7021 ggtgtaccac cagagtccgg gccaagcccc atacccgggt ccacagcagc caagctaccc
     7081 gttcccgcag ccaccacagc aaagttacta cccgcagcaa catcaccatc accatcatta
     7141 aggatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     7201 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     7261 gaggaactat atccggatat cccgcaagag gcccggcagt accggcataa ccaagcctat
     7321 gcctacagca tccagggtga cggtgccgag gatgacgatg agcgcattgt tagatttcat
     7381 acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt aaagcttatc
     7441 gatgataagc tgtcaaacat gagaattctt gaagacgaaa gggcctcgtg atacgcctat
     7501 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     7561 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     7621 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     7681 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     7741 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     7801 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     7861 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg
     7921 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     7981 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     8041 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     8101 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     8161 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     8221 caatggcaac aacgttgcgc aaactattaa ctggcgaact act
//
