LOCUS       sequence-372584-        6133 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon-optimized bacterial expression plasmid. Expresses ubiquitin 
            conjugating enzyme His-Tev-UbE2D3..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6133)
  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 6133)
  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-UbE2D3
FEATURES             Location/Qualifiers
     source          1..6133
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     32..49
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    64..203
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(129..148)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     289..311
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(305..496)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1808..2890)
                     /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     2852..2871
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2891..2968)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3174..3193
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3277..3296
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3277..3295
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3296..3320
                     /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             3335..3357
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3368..3385
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3386..3406
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3910..3928)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3924..3971
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4146..4151,4152..4168,4169..4174))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4201..4219)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4287..4391
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4392..4460,4461..5252)
                     /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(4610..4629)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5423..6011
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5912..5931
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
       61 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
      121 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
      181 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
      241 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
      301 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
      361 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
      421 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
      481 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
      541 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
      601 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
      661 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
      721 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
      781 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
      841 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
      901 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
      961 gtggccagga cccaacgctg cccgagatgc gccgcgtgcg gctgctggag atggcggacg
     1021 cgatggatat gttctgccaa gggttggttt gcgcattcac agttctccgc aagaattgat
     1081 tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt
     1141 cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa ggtatagggc
     1201 ggcgcctaca atccatgcca acccgttcca tgtgctcgcc gaggcggcat aaatcgccgt
     1261 gacgatcagc ggtccaatga tcgaagttag gctggtaaga gccgcgagcg atccttgaag
     1321 ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg cgggcatccc
     1381 gatgccgccg gaagcgagaa gaatcataat ggggaaggcc atccagcctc gcgtcgcgaa
     1441 cgccagcaag acgtagccca gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc
     1501 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     1561 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     1621 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     1681 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     1741 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     1801 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     1861 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     1921 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     1981 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     2041 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     2101 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     2161 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     2221 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     2281 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     2341 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     2401 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     2461 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     2521 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     2581 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     2641 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     2701 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     2761 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     2821 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     2881 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     2941 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     3001 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     3061 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     3121 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     3181 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     3241 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
     3301 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
     3361 acatatgcat catcatcatc atcatgaaaa cctgtatttt caaggcagcg gcagcatggc
     3421 gctgaaacgc attaacaaag aactgagcga tctggcgcgc gatccgccgg cgcagtgcag
     3481 cgcgggcccg gtgggcgatg atatgtttca ttggcaagcg accattatgg gcccgaacga
     3541 tagcccgtat caaggcggcg tgttttttct gaccattcat tttccgaccg attatccgtt
     3601 taaaccgccg aaagtggcgt ttaccacccg catttatcat ccgaacatta acagcaacgg
     3661 cagcatttgc ctggatattc tgcgcagtca gtggagcccg gcgctgacca ttagcaaagt
     3721 gctgctgagc atttgcagcc tgctgtgcga tccgaacccg gatgatccgc tggtgccgga
     3781 aattgcgcgc atttataaaa ccgatcgcga taaatataac cgcattagcc gcgaatggac
     3841 gcagaaatat gcgatgtaag gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     3901 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     3961 ggggtttttt gctgaaagga ggaactatat ccggatatcc cgcaagaggc ccggcagtac
     4021 cggcataacc aagcctatgc ctacagcatc cagggtgacg gtgccgagga tgacgatgag
     4081 cgcattgtta gatttcatac acggtgcctg actgcgttag caatttaact gtgataaact
     4141 accgcattaa agcttatcga tgataagctg tcaaacatga gaattcttga agacgaaagg
     4201 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     4261 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     4321 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     4381 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     4441 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     4501 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     4561 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     4621 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     4681 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     4741 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     4801 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     4861 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     4921 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     4981 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     5041 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     5101 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     5161 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     5221 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     5281 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     5341 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5401 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5461 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5521 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     5581 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     5641 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5701 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5761 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5821 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5881 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5941 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     6001 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     6061 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     6121 ttgagtgagc tga
//
