LOCUS       sequence-307560-        5231 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Bacterial expression plasmid for SARS-CoV-2 M (membrane).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5231)
  TITLE     SARS-CoV-2 protein E. coli expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 5231)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pGBW-m4136462
FEATURES             Location/Qualifiers
     source          1..5231
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..39
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    39..63
                     /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             78..100
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             111..128
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             141..1238
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1095..1104
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1212..1235
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1251..1271
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     terminator      2085..2116
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        2117..2219
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             join(2220..2288,2289..3080)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESLRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLATIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGSLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2438..2457)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             3081..3272
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     terminator      3296..3323
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(3335..3922)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3415..3434)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3944..3973)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     CDS             complement(4059..5141)
                     /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     5103..5122
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
       61 tcccctctag aaataatttt gtttaacttt aagaaggaga ctcttctatg caccatcatc
      121 accatcatgg tggttctggt aaaatcgaag aaggtaaact ggtaatctgg attaacggcg
      181 ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat accggaatta
      241 aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt gcggcaactg
      301 gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac gctcaatctg
      361 gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat ccgtttacct
      421 gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt gaagcgttat
      481 cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa gagatcccgg
      541 cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac
      601 cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag tatgaaaacg
      661 gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct
      721 tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac tccatcgcag
      781 aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca
      841 acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc aagggtcaac
      901 catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt ccgaacaaag
      961 agctggcgaa agagttcctc gaaaactatc tgctgactga tgaaggtctg gaagcggtta
     1021 ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag ttggcgaaag
     1081 atccacgtat tgccgccacc atggaaaacg cccagaaagg tgaaatcatg ccgaacatcc
     1141 cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc gccagcggtc
     1201 gtcagactgt cgatgaagcc ctgaaagacg cgcagactgg tggttctggt gagaacctgt
     1261 actttcaaag cgccgattca aacggtacga ttactgtaga agagcttaaa aagctgttag
     1321 aacaatggaa tttggtgatc gggtttctgt tcctgacctg gatctgtctc ctgcaattcg
     1381 cttacgcgaa ccgcaaccgg tttctgtata taatcaaatt aattttcctg tggctgctgt
     1441 ggcccgtcac cctggcatgc ttcgttctgg ctgctgttta cagaatcaat tggattactg
     1501 gcggtatcgc aatcgcgatg gcatgcctag taggcctgat gtggctgtcg tattttattg
     1561 cttccttccg tctgtttgcc cgtacccgta gcatgtggtc tttcaaccca gaaactaaca
     1621 tcctgcttaa cgttcctctg cacggtacaa tcctgacccg cccgctgctg gagagcgaac
     1681 tggtgattgg cgctgtcatc ttacgtggtc atctgcgtat tgctggccac catttgggtc
     1741 gctgtgacat caaagacttg ccgaaagaaa tcactgttgc aacctctcgt acactgtcct
     1801 actacaaact gggggcgagt cagcgtgttg ccggcgattc tggattcgcg gcttacagcc
     1861 gctatcgcat cggtaattac aagctcaaca ctgaccacag ctcctctagt gataacattg
     1921 cactgctggt gcagtaaaga gaatataaaa agccagatta ttaatccggc ttttttatta
     1981 tttaggttct gttaagtaac tgaacccaat gtcgttagtg acgcttacct cttaagaggt
     2041 cactgaccta acagcttgac tccagcgtaa ctggactgca atcaactcaa aagcctccga
     2101 ccggaggctt ttgacttgat cggcacgtaa gaggttccaa ctttcaccat aatgaaataa
     2161 gatcactacc gggcgtattt tttgagttat cgagattttc aggagctaag gaagctaaaa
     2221 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     2281 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     2341 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt ttacgccccg
     2401 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     2461 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     2521 ttgagtactc accagtcaca gaaaagcatc tcacggatgg catgacagta agagaattat
     2581 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg gcaacgatcg
     2641 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     2701 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     2761 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     2821 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggatca cttctgcgct
     2881 cggccctccc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     2941 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgcatcgta gttatctaca
     3001 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     3061 cactgattaa gcattggtaa gtgaccaaac aggaaaaaac cgcccttaac atggcccgct
     3121 ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac gcggatgaac
     3181 aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc agctgcctcg
     3241 cgcgtttcgg tgatgacggt gaaaacctct gatgagggcc caaatgtaat cacctggctc
     3301 accttcgggt gggcctttct gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3361 agcatcacaa aaatcgatgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3421 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3481 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3541 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3601 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3661 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     3721 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     3781 tatttggtat ctgcgctctg ctgaagccag ttacctcgga aaaagagttg gtagctcttg
     3841 atccggcaaa caaaccaccg ctggtagcgg tggtcttttt gtttgcaagc agcagattac
     3901 gcgcagaaaa aaaggatctc aagaagatcc tttgattttc taccgaagaa aggcccaccc
     3961 gtgaaggtga gccagtgact ttctgctatg gaggtcaggt atgatttaaa tggtcagtgc
     4021 ggcgcgccaa caaaaaaccc ctagcccccc gttttatatc actgcccgct ttccagtcgg
     4081 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4141 gtattgggcg ccagggtggt ttttcttttc accagtgaga ctggcaacag ctgattgccc
     4201 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     4261 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctatc ttcggtatcg
     4321 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     4381 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     4441 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     4501 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     4561 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     4621 agatgctcca cgcccagtcg cgtaccgtcc tcatgggaga aaataatact gttgatgggt
     4681 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     4741 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     4801 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     4861 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     4921 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     4981 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     5041 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     5101 ccggcatact ctgcgacatc gtataacgtt actggtttca tattcaccac cctgaattga
     5161 ctgtcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatactagta
     5221 gcggccggat g
//
