LOCUS       sequence-372590-        6079 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon-optimized bacterial expression plasmid. Expresses 
            Alpha-Synuclein-His..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6079)
  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 6079)
  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: Alpha-Synuclein-His
FEATURES             Location/Qualifiers
     source          1..6079
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(5..10,11..27,28..33))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(60..78)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        146..250
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(251..319,320..1111)
                     /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(469..488)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1282..1870
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1771..1790
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2024..2041
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2056..2195
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2121..2140)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2281..2303
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2297..2488)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3800..4882)
                     /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     4844..4863
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4883..4960)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5166..5185
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5269..5288
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5269..5287
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5288..5312
                     /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             5327..5349
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5777..5794
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5848..5866)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5862..5909
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ccgcattaaa gcttatcgat gataagctgt caaacatgag aattcttgaa gacgaaaggg
       61 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
      121 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
      181 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
      241 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
      301 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
      361 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
      421 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
      481 ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
      541 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
      601 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
      661 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
      721 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
      781 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
      841 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
      901 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
      961 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     1021 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     1081 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     1141 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     1201 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     1261 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     1321 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     1381 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     1441 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     1501 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     1561 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     1621 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     1681 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     1741 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     1801 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     1861 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     1921 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     1981 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     2041 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     2101 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     2161 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     2221 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     2281 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     2341 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     2401 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     2461 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     2521 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     2581 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     2641 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     2701 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     2761 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     2821 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     2881 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     2941 tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg cggctgctgg
     3001 agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc acagttctcc
     3061 gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg tgccgccggc
     3121 ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac
     3181 aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg ccgaggcggc
     3241 ataaatcgcc gtgacgatca gcggtccaat gatcgaagtt aggctggtaa gagccgcgag
     3301 cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca tggcctgcaa
     3361 cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg ccatccagcc
     3421 tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc cggcgataat
     3481 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     3541 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     3601 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     3661 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     3721 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     3781 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3841 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     3901 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     3961 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     4021 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     4081 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     4141 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     4201 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     4261 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     4321 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     4381 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     4441 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     4501 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     4561 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     4621 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     4681 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     4741 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     4801 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     4861 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     4921 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     4981 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     5041 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     5101 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     5161 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     5221 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca
     5281 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta
     5341 agaaggagat atacatatgg atgtgtttat gaaaggcctg agcaaagcga aagaaggtgt
     5401 agtcgcggcg gcggaaaaaa ccaaacaagg cgtggcggaa gcggcgggaa aaaccaagga
     5461 aggagtttta tatgtgggca gcaaaaccaa agagggagta gtccatggcg tggcgaccgt
     5521 ggcggagaaa acgaaggaac aagtgaccaa cgtgggcggc gcggtggtga ccggcgtgac
     5581 cgcggtggcg cagaaaaccg tggaaggcgc gggcagcatt gcggcggcga ccggctttgt
     5641 gaaaaaagat cagctgggca aaaacgaaga aggcgcgccg caagaaggca ttctggaaga
     5701 tatgccggtt gatccggata acgaagcgta tgaaatgccg agcgaagaag gctatcaaga
     5761 ttatgaaccg gaagcgcatc atcatcatca tcattaagga tccggctgct aacaaagccc
     5821 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     5881 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggatatcccg
     5941 caagaggccc ggcagtaccg gcataaccaa gcctatgcct acagcatcca gggtgacggt
     6001 gccgaggatg acgatgagcg cattgttaga tttcatacac ggtgcctgac tgcgttagca
     6061 atttaactgt gataaacta
//
