LOCUS       sequence-372589-        8293 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon-optimized bacterial expression plasmid. Expresses ubiquitin 
            ligating enzyme His-Tev-WWP2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8293)
  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 8293)
  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: His-Tev-WWP2
FEATURES             Location/Qualifiers
     source          1..8293
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     89..111
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(105..296)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1608..2690)
                     /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     2652..2671
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2691..2768)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2974..2993
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3077..3096
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3077..3095
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3096..3120
                     /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             3135..3157
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3168..3185
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3186..3206
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(5870..5888)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5884..5931
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6106..6111,6112..6128,6129..6134))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6161..6179)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6247..6351
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6352..6420,6421..7212)
                     /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(6570..6589)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7383..7971
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7872..7891
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8125..8142
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(8157..8293,1..3)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8222..8241)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
       61 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
      121 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
      181 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
      241 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
      301 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
      361 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
      421 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
      481 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
      541 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
      601 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
      661 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
      721 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg
      781 cccgagatgc gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa
      841 gggttggttt gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg
      901 gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg
      961 caccgcgacg caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca
     1021 acccgttcca tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccaatga
     1081 tcgaagttag gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat
     1141 ctacctgcct ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa
     1201 gaatcataat ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca
     1261 gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     1321 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     1381 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     1441 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     1501 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     1561 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     1621 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     1681 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     1741 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     1801 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     1861 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     1921 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     1981 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     2041 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     2101 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     2161 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     2221 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     2281 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     2341 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     2401 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     2461 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     2521 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     2581 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     2641 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     2701 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     2761 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     2821 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     2881 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     2941 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     3001 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
     3061 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
     3121 cctctagaaa taattttgtt taactttaag aaggagatat acatatgcat catcatcatc
     3181 atcatgaaaa cctgtatttt caaggcatgg cgagcgcgag cagcagccgc gcgggcgtgg
     3241 cgctgccgtt tgaaaaaagt cagctaaccc tgaaggtggt gagcgcgaaa ccgaaagtgc
     3301 ataaccgtca gccgcgcatt aacagctatg tggaagtggc ggtggatggc ctgccgagcg
     3361 aaaccaaaaa aaccggcaaa cgcattggca gcagcgaact gctgtggaac gaaattatta
     3421 ttctgaacgt gaccgcgcag agccatctgg atctgaaagt gtggagctgc cataccctgc
     3481 gcaacgaact gctgggcacc gcgagcgtga acctgagcaa cgtgctgaaa aacaacggcg
     3541 gcaaaatgga aaacatgcag ctgaccctga atttacagac cgaaaacaaa ggcagcgtgg
     3601 tgagcggcgg cgaactgacc atttttctgg atggccctac cgtggatctg ggcaatgtgc
     3661 cgaacggctc agccttaacc gatggtagtc agctgccgag cagagatagc tctggtaccg
     3721 cggtggcgcc ggaaaaccgc catcagccgc cgagcaccaa ctgcttcggc ggccggagca
     3781 gaacccatag gcatagcggc gcatcggcgc gaacaacccc tgcaacgggg gaacaatccc
     3841 cgggggcgcg gtctcgccat cgtcagccgg tgaaaaacag cggccatagc ggcctggcga
     3901 acggcaccgt gaacgatgaa ccgaccaccg cgaccgatcc ggaggaacct agcgtagtcg
     3961 gcgtcacgag cccgccggcc gcgcctttaa gcgtgacccc aaacccgaat acgacgagct
     4021 taccggctcc ggcgacaccg gcggagggcg aagaaccgag cacctctggt acccaacagc
     4081 tccctgcggc ggcccaagcg cccgacgcgc tgcccgcggg ctgggaacag cgcgaactgc
     4141 cgaacggccg cgtgtattac gtggatcaca acaccaaaac caccacctgg gaacgtccgc
     4201 tgccgcccgg ttgggaaaag agaaccgatc cgcgcggccg cttttattac gtggaccaca
     4261 atacacgaac tacaacctgg cagcgcccga ccgcggaata tgtgcgcaac tatgaacagt
     4321 ggcagagtca gcgcaatcag ctgcaaggcg cgatgcagca ttttagtcag cgctttctgt
     4381 atcagagcag cagcgcgagc accgatcatg atccgctggg ccctcttccc ccgggctggg
     4441 agaaacgcca agataatggc cgtgtgtact acgtgaatca caacacccgc accacgcagt
     4501 gggaagatcc gcgcacccaa ggcatgattc aagaaccggc actgcctccg gggtgggaaa
     4561 tgaaatatac gagcgaaggc gttcgctatt ttgtagacca taacacgcga accacaacgt
     4621 ttaaagatcc gcgcccgggc tttgaaagcg gcaccaaaca aggcagtccg ggcgcgtacg
     4681 atcgcagctt tcgctggaaa tatcatcagt ttcgctttct gtgccatagc aacgcgctgc
     4741 cgagccatgt gaaaattagc gtgagccgtc agaccctgtt tgaagatagc tttcagcaga
     4801 ttatgaacat gaaaccgtat gatctgcgcc gccgcctgta tattattatg cgcggcgaag
     4861 aaggcctgga ttatggcggc attgcgcgcg aatggttttt tctgctgagc catgaagtgc
     4921 tgaacccgat gtattgcctg tttgaatatg cgggcaaaaa caactattgc ctgcagatta
     4981 acccggcgag cagcattaac ccggatcatc tgacctattt tcgctttatt ggccgcttta
     5041 ttgcgatggc gctgtatcat ggcaaattta ttgataccgg ctttaccctg ccgttttata
     5101 aacgcatgct gaacaaacgc ccgaccctga aagatctgga aagcattgat ccggaatttt
     5161 ataacagcat tgtgtggatt aaagaaaaca acctggaaga atgcggcctg gaactgtatt
     5221 ttattcaaga tatggaaatt ctgggcaaag tgaccaccca tgaactgaaa gaaggcggcg
     5281 aaagcattcg cgtgaccgaa gaaaacaaag aagaatatat tatgctgctg accgattggc
     5341 gctttacccg cggcgtggaa gaacagacca aagcgttttt agatggcttc aacgaagtgg
     5401 cgccgctgga atggctgcgc tattttgacg aaaaagaact ggaactgatg ctgtgcggca
     5461 tgcaagaaat tgatatgagc gattggcaga aaagcaccat ttatcgccat tataccaaaa
     5521 acagcaaaca gattcagtgg ttttggcaag tggtgaaaga aatggataac gaaaaacgca
     5581 ttcgcctgct gcagtttgtg accggcacct gccgcctgcc ggtgggcggc tttgcggaac
     5641 tgattggcag caacggcccg cagaaatttt gcattgataa agtgggcaaa gaaacctggc
     5701 tgccgcgcag ccatacctgc tttaaccgcc tggatctgcc gccgtataaa agctatgaac
     5761 agctgcgcga aaaactgctg tatgcgattg aagaaaccga aggctttggc caagaataag
     5821 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     5881 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     5941 ggaactatat ccggatatcc cgcaagaggc ccggcagtac cggcataacc aagcctatgc
     6001 ctacagcatc cagggtgacg gtgccgagga tgacgatgag cgcattgtta gatttcatac
     6061 acggtgcctg actgcgttag caatttaact gtgataaact accgcattaa agcttatcga
     6121 tgataagctg tcaaacatga gaattcttga agacgaaagg gcctcgtgat acgcctattt
     6181 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     6241 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     6301 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     6361 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     6421 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     6481 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     6541 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac
     6601 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     6661 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     6721 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     6781 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     6841 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca
     6901 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     6961 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     7021 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     7081 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     7141 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     7201 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     7261 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     7321 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     7381 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     7441 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     7501 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     7561 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     7621 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     7681 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     7741 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     7801 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     7861 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     7921 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     7981 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     8041 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     8101 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     8161 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     8221 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     8281 ctgggtcatg gct
//
