LOCUS       sequence-372587-        8857 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Codon-optimized bacterial expression plasmid. Expresses ubiquitin 
            activating enzyme His-Tev-UbE1..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8857)
  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 8857)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-UbE1
FEATURES             Location/Qualifiers
     source          1..8857
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     66..88
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(82..273)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1585..2667)
                     /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     2629..2648
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2668..2745)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2951..2970
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3054..3073
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3054..3072
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3073..3097
                     /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             3112..3134
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3145..3162
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3163..3183
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      6425..6472
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6647..6652,6653..6669,6670..6675))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6702..6720)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6788..6892
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6893..6961,6962..7753)
                     /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(7111..7130)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7924..8512
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8413..8432
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8666..8683
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8698..8837
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8763..8782)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
       61 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
      121 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
      181 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
      241 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
      301 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
      361 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
      421 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
      481 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
      541 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
      601 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
      661 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
      721 cacgatcatg cgcacccgtg gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct
      781 gctggagatg gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg cattcacagt
      841 tctccgcaag aattgattgg ctccaattct tggagtggtg aatccgttag cgaggtgccg
      901 ccggcttcca ttcaggtcga ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg
      961 cagacaaggt atagggcggc gcctacaatc catgccaacc cgttccatgt gctcgccgag
     1021 gcggcataaa tcgccgtgac gatcagcggt ccaatgatcg aagttaggct ggtaagagcc
     1081 gcgagcgatc cttgaagctg tccctgatgg tcgtcatcta cctgcctgga cagcatggcc
     1141 tgcaacgcgg gcatcccgat gccgccggaa gcgagaagaa tcataatggg gaaggccatc
     1201 cagcctcgcg tcgcgaacgc cagcaagacg tagcccagcg cgtcggccgc catgccggcg
     1261 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     1321 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     1381 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     1441 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     1501 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     1561 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     1621 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     1681 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     1741 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     1801 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     1861 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     1921 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     1981 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     2041 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     2101 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     2161 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     2221 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     2281 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     2341 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     2401 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     2461 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     2521 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     2581 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     2641 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     2701 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     2761 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     2821 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     2881 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     2941 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     3001 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
     3061 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
     3121 ctttaagaag gagatataca tatgcatcat catcatcatc atgaaaacct gtattttcaa
     3181 ggcatgagca gcagcccgct gagcaaaaaa cgccgcgtga gcggccctga tccgaaaccg
     3241 ggcagcaact gcagcccggc gcagagcgtg ctgagcgaag tgccgagcgt gccgaccaac
     3301 ggaatggcga aaaacggcag cgaagcggat attgatgaag gcctgtatag ccgtcagctg
     3361 tatgtgctgg gccatgaagc gatgaaacgc ctgcagacga gcagcgtgct ggtgagcggc
     3421 ctgcgcggcc tgggcgtgga aattgcgaaa aacattattc tgggcggcgt gaaagcggtg
     3481 accctgcatg atcaaggcac cgcgcagtgg gcggatctga gcagtcagtt ttatctgcgc
     3541 gaagaagata ttggcaaaaa ccgcgcggaa gtgagtcagc cgcgcttagc ggaactgaac
     3601 agctatgtgc cggtgaccgc gtataccggc ccgctggtgg aagattttct gagcggcttt
     3661 caagtggtgg tgctgaccaa caccccgctg gaagatcagc tgcgcgtggg cgaattttgc
     3721 cataaccgcg gcattaaact ggtggtggcg gatacccgcg gcctgtttgg tcagctgttt
     3781 tgcgattttg gagaagaaat gattctgacc gatagcaacg gcgaacagcc gctgagcgcg
     3841 atggtgagca tggtgaccaa agataacccg ggcgtggtga cctgcctgga tgaagcgcgc
     3901 catggctttg agagcggaga tttcgtgagc tttagcgagg tacaaggcat ggtcgaactg
     3961 aacgggaatc agccgatgga aattaaagtg ctgggcccgt atacctttag catttgcgat
     4021 acgagcaact ttagcgatta tattcgcggc ggcattgtga gccaagtgaa agtgccgaaa
     4081 aaaattagct ttaaaagcct ggtggcgagc ctggcggaac cggattttgt ggtgaccgat
     4141 tttgcgaaat ttagccgccc ggcgcagctg catattggct ttcaagcgtt acatcagttt
     4201 tgtgctcaac acggccgccc gccgcgccct cgcaacgaag aagatgcagc ggagctggtg
     4261 gcgctggcac aagcggtgaa cgctcgcgcg ctgccggcgg tgcagcagaa caacctggat
     4321 gaagatctga ttcgcaaact ggcgtatgtg gcggcgggcg atctggcgcc gattaacgcg
     4381 tttattggcg gcctggcggc gcaagaagtg atgaaagcgt gcagcggcaa atttatgccg
     4441 attatgcagt ggctgtattt tgatgcgctg gaatgcctgc cggaagataa agaagtgctg
     4501 accgaagata aatgcctgca gaggcagaat cgctatgatg gccaagtggc ggtgtttggc
     4561 agcgatctgc aagaaaaact gggcaaacag aaatattttc tggtgggcgc gggcgcgatt
     4621 ggctgcgaac tgctgaaaaa ctttgcaatg attggcctgg gctgcggcga aggcggcgaa
     4681 attattgtga ccgatatgga taccattgaa aaaagcaacc tgaaccgtca gtttctgttt
     4741 cgcccgtggg atgtgaccaa actgaaaagc gataccgcgg cggcggcggt gcgtcagatg
     4801 aacccgcata ttcgcgtgac gagccatcag aaccgcgtgg gcccggatac cgaacgcatt
     4861 tatgatgatg atttttttca gaacctggat ggcgtggcga acgcgctgga taacgtggat
     4921 gcgcgcatgt atatggatcg ccgctgcgtg tattatcgca aaccgctgct ggaaagcggc
     4981 accctgggca ccaaaggcaa cgtgcaagtg gtgattccgt ttctgaccga aagctatagc
     5041 agttcgcaag atcctccgga aaaaagcatt ccgatttgca ccctgaagaa ttttccaaac
     5101 gcgattgaac ataccctgca gtgggcgcgc gatgaatttg aaggcctgtt taaacagccg
     5161 gcggaaaacg tgaatcagta tctgaccgat ccgaaatttg tggaacgcac cctgcgcctg
     5221 gcgggcacgc agccgctgga agtgctggaa gcggtgcagc gcagcctggt gctgcaacgc
     5281 ccgcagacct gggcggattg cgtgacctgg gcgtgccatc attggcatac gcagtatagc
     5341 aacaacattc gtcagctgct gcataacttt ccgccggatc agctgacgag cagcggcgcg
     5401 ccgttttgga gcggcccgaa acgctgcccg catccgctga cctttgatgt gaacaacccg
     5461 ctgcatctgg attatgtgat ggcggcggcg aacctgtttg cacaaaccta tggcctgact
     5521 ggcagccaag accgcgctgc ggtggcgacc tttctgcaga gcgtgcaagt gccggaattt
     5581 accccgaaaa gcggcgtgaa aattcatgtg agcgatcaag aactgcagag cgcgaacgcg
     5641 agcgtggatg atagccgcct ggaagaactg aaagcgaccc tgccgagccc ggataaactg
     5701 ccgggcttta aaatgtatcc gattgatttt gaaaaagatg atgatagcaa ctttcacatg
     5761 gattttattg tggcggcgag caacctgcgc gcggaaaact atgatattcc gagcgcggat
     5821 cgccataaaa gcaaactgat tgcgggcaaa attattccgg cgattgcgac caccaccgcg
     5881 gcggtggtgg gcctggtgtg cctggaactg tataaagtgg tgcaaggcca tcgtcagctg
     5941 gatagctata aaaacggctt tctgaacctg gcgctgccgt tttttggctt tagcgaaccg
     6001 ctggcggcgc cgcgccatca gtattataac caagaatgga ccctgtggga tcgctttgaa
     6061 gtacaaggcc tgcagccgaa cggtgaagaa atgaccctga aacagtttct ggattatttt
     6121 aaaaccgaac ataaactgga aattaccatg ctgagccaag gcgtgagcat gctgtatagc
     6181 ttttttatgc cggcggcgaa actgaaagaa cgcctggatc agccgatgac cgaaattgtg
     6241 agccgcgtga gcaaacgcaa actgggccgc catgtgcgcg cgctggtgct ggaactgtgc
     6301 tgcaacgatg agagcggcga agatgtggaa gtgccgtatg tgcgctatac cattcgctaa
     6361 ggatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     6421 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     6481 aggaactata tccggatatc ccgcaagagg cccggcagta ccggcataac caagcctatg
     6541 cctacagcat ccagggtgac ggtgccgagg atgacgatga gcgcattgtt agatttcata
     6601 cacggtgcct gactgcgtta gcaatttaac tgtgataaac taccgcatta aagcttatcg
     6661 atgataagct gtcaaacatg agaattcttg aagacgaaag ggcctcgtga tacgcctatt
     6721 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     6781 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     6841 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     6901 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     6961 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     7021 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     7081 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga
     7141 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     7201 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     7261 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     7321 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     7381 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc
     7441 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     7501 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     7561 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     7621 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     7681 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     7741 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     7801 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     7861 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     7921 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     7981 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     8041 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     8101 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     8161 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     8221 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     8281 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     8341 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     8401 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     8461 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     8521 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     8581 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     8641 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     8701 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
     8761 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     8821 actgggtcat ggctgcgccc cgacacccgc caacacc
//
