LOCUS       sequence-372592-        6001 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon-optimized bacterial expression plasmid. Expresses 
            Twin-Strep(R)-Tev-GSGS-Ubiquitin..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6001)
  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 6001)
  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: Twin-Strep(R)-Tev-GSGS-Ubiquitin
FEATURES             Location/Qualifiers
     source          1..6001
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      505..1093
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     994..1013
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1247..1264
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1279..1418
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1344..1363)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1504..1526
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1520..1711)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3023..4105)
                     /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     4067..4086
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4106..4183)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4389..4408
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4492..4511
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4492..4510
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4511..4535
                     /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             4550..4572
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4682..4702
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4712..4939
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     primer_bind     complement(4993..5011)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5007..5054
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5229..5234,5235..5251,5252..5257))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5284..5302)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5370..5474
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5475..5543,5544..6001,1..334)
                     /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(5693..5712)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact
       61 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
      121 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
      181 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
      241 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
      301 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
      361 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
      421 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
      481 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
      541 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
      601 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
      661 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
      721 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
      781 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
      841 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
      901 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
      961 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     1021 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     1081 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     1141 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     1201 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     1261 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     1321 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     1381 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     1441 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     1501 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     1561 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     1621 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     1681 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     1741 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     1801 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     1861 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     1921 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     1981 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     2041 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     2101 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     2161 cgatcatgcg cacccgtggc caggacccaa cgctgcccga gatgcgccgc gtgcggctgc
     2221 tggagatggc ggacgcgatg gatatgttct gccaagggtt ggtttgcgca ttcacagttc
     2281 tccgcaagaa ttgattggct ccaattcttg gagtggtgaa tccgttagcg aggtgccgcc
     2341 ggcttccatt caggtcgagg tggcccggct ccatgcaccg cgacgcaacg cggggaggca
     2401 gacaaggtat agggcggcgc ctacaatcca tgccaacccg ttccatgtgc tcgccgaggc
     2461 ggcataaatc gccgtgacga tcagcggtcc aatgatcgaa gttaggctgg taagagccgc
     2521 gagcgatcct tgaagctgtc cctgatggtc gtcatctacc tgcctggaca gcatggcctg
     2581 caacgcgggc atcccgatgc cgccggaagc gagaagaatc ataatgggga aggccatcca
     2641 gcctcgcgtc gcgaacgcca gcaagacgta gcccagcgcg tcggccgcca tgccggcgat
     2701 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     2761 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     2821 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     2881 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     2941 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     3001 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     3061 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     3121 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     3181 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     3241 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     3301 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     3361 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     3421 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     3481 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     3541 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     3601 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     3661 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     3721 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     3781 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     3841 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     3901 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     3961 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     4021 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     4081 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     4141 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     4201 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     4261 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     4321 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     4381 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     4441 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac
     4501 tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact
     4561 ttaagaagga gatatacata tggggagctg gagccacccg cagtttgaaa gaggcggcgg
     4621 tagcggagga gggtcgggag gcagcagcgc gtggagccat ccgcagtttg aacgcggcag
     4681 cgaaaacctg tattttcaag gcagcggcag catgcagatt tttgtgaaaa ccctgaccgg
     4741 caaaaccatt accctggaag tggaaccgag cgataccatt gaaaacgtga aagcgaaaat
     4801 tcaagataaa gaaggcattc cgccggatca gcagcgcctg atttttgcgg gcaaacagct
     4861 ggaagatggc cgcaccctga gcgattataa cattcagaaa gaaagcaccc tgcatctggt
     4921 gctgcgcctg cgcggcggct aaggatccgg ctgctaacaa agcccgaaag gaagctgagt
     4981 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     5041 tgaggggttt tttgctgaaa ggaggaacta tatccggata tcccgcaaga ggcccggcag
     5101 taccggcata accaagccta tgcctacagc atccagggtg acggtgccga ggatgacgat
     5161 gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta actgtgataa
     5221 actaccgcat taaagcttat cgatgataag ctgtcaaaca tgagaattct tgaagacgaa
     5281 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     5341 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5401 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5461 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5521 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5581 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5641 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5701 gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5761 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5821 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5881 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5941 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     6001 g
//
