LOCUS       sequence-372586-        8608 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon-optimized bacterial expression plasmid. Expresses ubiquitin 
            ligating enzyme His-Tev-Nedd4L..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8608)
  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 8608)
  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-Nedd4L
FEATURES             Location/Qualifiers
     source          1..8608
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(995..2077)
                     /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     2039..2058
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2078..2155)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2361..2380
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2464..2483
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2464..2482
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2483..2507
                     /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             2522..2544
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2555..2572
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2573..2593
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(5572..5590)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5586..5633
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5808..5813,5814..5830,5831..5836))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5863..5881)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5949..6053
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6054..6122,6123..6914)
                     /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(6272..6291)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7085..7673
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7574..7593
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7827..7844
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7859..7998
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7924..7943)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8084..8106
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(8100..8291)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
       61 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
      121 acgacaggag cacgatcatg cgcacccgtg gccaggaccc aacgctgccc gagatgcgcc
      181 gcgtgcggct gctggagatg gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg
      241 cattcacagt tctccgcaag aattgattgg ctccaattct tggagtggtg aatccgttag
      301 cgaggtgccg ccggcttcca ttcaggtcga ggtggcccgg ctccatgcac cgcgacgcaa
      361 cgcggggagg cagacaaggt atagggcggc gcctacaatc catgccaacc cgttccatgt
      421 gctcgccgag gcggcataaa tcgccgtgac gatcagcggt ccaatgatcg aagttaggct
      481 ggtaagagcc gcgagcgatc cttgaagctg tccctgatgg tcgtcatcta cctgcctgga
      541 cagcatggcc tgcaacgcgg gcatcccgat gccgccggaa gcgagaagaa tcataatggg
      601 gaaggccatc cagcctcgcg tcgcgaacgc cagcaagacg tagcccagcg cgtcggccgc
      661 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
      721 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
      781 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
      841 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
      901 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
      961 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     1021 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     1081 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     1141 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     1201 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     1261 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     1321 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     1381 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     1441 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     1501 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     1561 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     1621 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     1681 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     1741 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     1801 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     1861 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     1921 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     1981 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     2041 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     2101 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     2161 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     2221 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     2281 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     2341 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     2401 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
     2461 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
     2521 ttttgtttaa ctttaagaag gagatataca tatgcatcat catcatcatc atgaaaacct
     2581 gtattttcaa ggcatggcga ccggcttagg cgaacccgtg tatggcctga gcgaagatga
     2641 aggcgaaagc cgcattctgc gcgtgaaagt ggtgagcggc attgatctgg cgaaaaaaga
     2701 tatttttggc gcgagcgatc cgtatgtgaa actgagcctg tatgtggcgg atgagaatcg
     2761 cgaactggcg ctggtgcaga ccaaaaccat taaaaaaacc ctcaatccga aatggaacga
     2821 agaattttat tttcgcgtga acccgagcaa ccatcgcctg ctgtttgaag tgtttgacga
     2881 aaacagattg acccgcgatg attttctggg ccaagtggat gtgccgctga gccatctgcc
     2941 gaccgaagat ccgaccatgg aacgcccgta tacctttaaa gattttctgc tgcgcccgag
     3001 gagccataag agccgcgtga aaggctttct gcgcctgaaa atggcgtata tgccgaaaaa
     3061 cggcggccaa gatgaagaga actcggatca gcgcgatgat atggaacatg gctgggaagt
     3121 ggtggatagc aacgatagcg cgagtcagca tcaagaagaa ctgcctccgc cgcccctgcc
     3181 tcctggctgg gaggagaaag tggataacct gggacgcacg tattatgtta accataacaa
     3241 ccgcaccacc caatggcatc gcccgagcct gatggatgtg agcagcgaaa gcgataacaa
     3301 cattcgtcag attaaccaag aagcggcgca tcgccgcttt cgcagccgcc gccatattag
     3361 cgaagatctg gaaccggaac cgagcgaagg cggcgatgtg ccggaaccgt gggaaaccat
     3421 tagcgaagaa gtgaatattg ccggcgattc tctggggtta gcgcttcccc caccccccgc
     3481 ttcaccgggc agccgcacga gcccgcaaga actgagcgaa gaactgagcc gccgcctgca
     3541 gattaccccg gatagcaacg gcgaacagtt tagcagcctg attcagcgcg aaccgagcag
     3601 ccgcctgcgc agctgtagcg taaccgatgc tgttgcagag caaggacatt tacctccgcc
     3661 gtcggcgccg gcgggtcggg cgagaagcag taccgtgacc ggcggcgaag aaccgacccc
     3721 gagcgtggcg tatgtgcata ccaccccggg cctgccgagc ggttgggaag agcgcaaaga
     3781 tgcgaaaggc cgcacctact atgtgaacca taacaatcgc acaaccactt ggacccgccc
     3841 gattatgcag ctggcggaag atggcgcgag cggcagcgcg accaacagca acaaccatct
     3901 gattgaaccg cagatacgcc gcccgcgctc gttgagctcg ccgaccgtga ccctgagcgc
     3961 gccgctggaa ggcgcgaaag atagcccggt gcgccgcgcg gtgaaagata ccctgagcaa
     4021 cccgcagagc ccgcagccga gcccgtataa cagcccgaaa ccgcagcata aagtgacgca
     4081 gagcttttta ccgccgggct gggagatgag aattgcgccg aacggccgcc cgttttttat
     4141 tgatcacaac accaaaacaa ccacctggga agatccgcgc ttgaaatttc cggtgcacat
     4201 gcgcagcaaa acgagcctga acccgaacga tctgggcccg cttccgccgg gctgggaaga
     4261 gcgcattcat ctggatggcc gcacctttta tatagaccat aattcaaaga ttacgcagtg
     4321 ggaagatccg cgattacaaa acccggcgat taccggcccg gcggtgccgt atagccgcga
     4381 atttaaacag aaatatgatt attttcgcaa aaaactgaaa aaaccggcgg atattccgaa
     4441 ccgctttgaa atgaaactgc atcgcaacaa catttttgaa gaaagctatc gccgcattat
     4501 gagcgtgaaa cgcccggatg tgctgaaagc gcgcctgtgg attgaatttg aaagcgaaaa
     4561 aggcctggat tatggcggcg tggcgcgcga atggtttttt ctgctgagca aagaaatgtt
     4621 taacccgtat tatggcctgt ttgaatatag cgcgaccgat aactataccc tgcagattaa
     4681 cccgaacagc ggcctgtgca acgaagatca tctgagctat tttaccttta ttggccgcgt
     4741 ggcgggcctg gcggtgtttc atggcaaact gctggatggc ttttttattc gcccgtttta
     4801 taaaatgatg ctgggcaaac agattaccct gaacgatatg gaaagcgtgg atagcgaata
     4861 ttataacagc ctgaaatgga ttctggaaaa cgatccgacc gaactggatc tgatgttttg
     4921 cattgatgag gaaaactttg gtcagaccta tcaagtggat ctgaaaccga acggcagcga
     4981 aattatggtg accaacgaaa acaaacgcga atatattgat ctggtgattc aatggcgctt
     5041 tgtgaaccgc gtgcagaaac agatgaacgc gtttctggaa ggctttaccg aactgctgcc
     5101 gattgatctg attaaaattt ttgatgaaaa cgagctggaa ctgctgatgt gcggcctggg
     5161 cgatgtggat gtgaacgatt ggcgtcagca tagcatttat aaaaacggct attgcccgaa
     5221 ccatccggtg attcagtggt tttggaaagc ggtgctgctg atggatgcgg aaaaacgcat
     5281 tcgcctgctg cagtttgtga ccggcacgag ccgcgtgccg atgaacggct ttgcggaact
     5341 gtatggcagc aacggcccgc agctgtttac cattgaacag tggggcagcc cggaaaaact
     5401 gcctcgtgcg cacacctgct ttaaccgcct ggatctgccg ccgtatgaaa cctttgaaga
     5461 tctgcgcgaa aaactgctga tggcggtgga aaacgcgcaa ggctttgaag gcgtggatta
     5521 aggatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     5581 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     5641 gaggaactat atccggatat cccgcaagag gcccggcagt accggcataa ccaagcctat
     5701 gcctacagca tccagggtga cggtgccgag gatgacgatg agcgcattgt tagatttcat
     5761 acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt aaagcttatc
     5821 gatgataagc tgtcaaacat gagaattctt gaagacgaaa gggcctcgtg atacgcctat
     5881 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     5941 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     6001 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     6061 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     6121 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     6181 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     6241 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg
     6301 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     6361 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     6421 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     6481 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     6541 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     6601 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6661 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6721 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     6781 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     6841 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     6901 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     6961 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     7021 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     7081 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     7141 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     7201 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     7261 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     7321 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     7381 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     7441 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     7501 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     7561 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     7621 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     7681 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     7741 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     7801 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     7861 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     7921 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     7981 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     8041 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     8101 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     8161 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     8221 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     8281 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     8341 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     8401 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     8461 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     8521 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     8581 ctttacgaaa cacggaaacc gaagacca
//
