LOCUS       sequence-311066-        6858 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Bacterial co-expression vector fo Covid-SARS2 NSP16 and NSP10.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6858)
  AUTHORS   Gileadi Lab
  TITLE     Bacterial co-expression vector for Covid-SARS2 NSP16 and NSP10
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6858)
  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: 
            "Unpublished"
COMMENT     Sequence Label: COVID-SARS2 NSP16/10
FEATURES             Location/Qualifiers
     source          1..6858
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     970..989
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1009..1086)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1292..1311
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1395..1414
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1395..1413
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1414..1438
                     /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             1453..1475
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1486..1503
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1513..1683
                     /codon_start=1
                     /product="Z domain of staphylococcal protein A, engineered 
                     to contain multiple positive charges (Hedhammar and Hober, 
                     2007)"
                     /label=Z-basic tag
                     /note="useful for purifying tagged proteins by 
                     cation-exchange chromatography"
                     /translation="VDNKFNKERRRARREIRHLPNLNREQRRAFIRSLRDDPSQSANLL
                     AEAKKLNDAQPK"
     CDS             1690..1710
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3117..3134
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3187..3205)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3201..3248
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3285..3740
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3372..3391)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3582..3603
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3832..4647)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3832..4647)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4555..4574
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4639..4658
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4769..5357
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5258..5277
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5511..5528
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5543..5682
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5608..5627)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5768..5790
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5784..5975)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6784..6858,1..1008))
                     /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"
ORIGIN
        1 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
       61 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
      121 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
      181 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
      241 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
      301 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
      361 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
      421 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
      481 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
      541 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
      601 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
      661 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
      721 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
      781 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
      841 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
      901 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
      961 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     1021 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     1081 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     1141 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     1201 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     1261 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     1321 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     1381 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     1441 tctagaaata attttgttta actttaagaa ggagatatac atatgcacca tcatcatcat
     1501 cattcttctg gtgtggataa caagttcaac aaggagcgtc gaagagctcg ccgtgaaatt
     1561 cgccatctgc cgaacctgaa ccgcgaacag cgtcgcgcat ttattcgcag cctgcgcgat
     1621 gatccgagcc agagcgcgaa cctgctggcg gaagcgaaga agctgaacga tgcgcagccg
     1681 aagggtaccg agaacctgta cttccaatcc atgagctccc aggcgtggca acccggcgtg
     1741 gccatgccga atctttacaa aatgcaacgc atgctcctgg aaaaatgcga cttacagaac
     1801 tacggcgata gcgcaacgct gccgaaaggt attatgatga acgtcgcgaa atatacgcag
     1861 ctgtgtcagt atctgaatac cctgaccctg gctgtcccgt ataacatgcg tgtcattcat
     1921 ttcggtgctg gcagtgataa aggcgtcgcg ccaggcacgg cagtgcttcg ccaatggtta
     1981 ccaactggta cattgctggt ggattcagat ctgaatgact ttgtttcgga tgcggatagc
     2041 acactgattg gtgattgtgc aactgtgcat acggccaata aatgggatct gattatctct
     2101 gacatgtatg acccaaaaac aaaaaacgta accaaagaaa acgatagtaa ggagggtttt
     2161 tttacctata tttgcggctt cattcagcag aaactggcac tcggcggcag cgtagccatt
     2221 aaaattaccg aacatagctg gaatgccgat ctgtacaagt taatgggcca ttttgcgtgg
     2281 tggacggcat tcgtaacgaa tgtgaacgcc tcaagttcag aagcgtttct catcgggtgc
     2341 aactatttag gtaaaccgcg cgaacaaatt gacggctacg taatgcatgc taattacatt
     2401 ttttggcgca acaccaatcc aattcagctg tcaagctatt cacttttcga tatgtcgaaa
     2461 tttccgctga aactgcgtgg aacggcggtg atgtcactga aagaaggaca aatcaacgat
     2521 atgattctct ccctgttatc aaaaggccgt ctgatcattc gggaaaacaa ccgcgtggtc
     2581 attagtagcg atgtactcgt aaataattaa taataattta aggagatata catggcggga
     2641 aacgcgaccg aagtgcctgc aaattctaca gtgctgtcct tctgtgcctt tgctgttgac
     2701 gcggcgaagg catacaaaga ctatctcgcg tctggaggcc agccgatcac aaattgcgtc
     2761 aaaatgctgt gtacacacac cggcacgggt caggcaatta ccgtaacccc ggaagccaac
     2821 atggatcagg aaagtttcgg cggagcaagc tgttgcctct attgtcgttg ccatatcgac
     2881 caccccaacc cgaaaggctt ttgcgattta aaaggcaagt atgtgcagat cccgacgacc
     2941 tgcgcgaacg atccggtggg cttcacgtta aaaaacaccg tgtgcaccgt gtgtggtatg
     3001 tggaaaggct atggctgtag ttgtgatcag ttacgcgagc caatgttgca gtaacagtaa
     3061 aggtggatac ggatccgaat tcgagctccg tcgacaagct tgcggccgca ctcgagcacc
     3121 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     3181 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     3241 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     3301 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3361 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3421 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3481 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3541 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3601 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3661 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     3721 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3781 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa
     3841 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     3901 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     3961 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     4021 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     4081 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     4141 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     4201 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     4261 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     4321 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     4381 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     4441 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     4501 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     4561 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca
     4621 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     4681 cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4741 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4801 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4861 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     4921 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     4981 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5041 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5101 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5161 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5221 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5281 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5341 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5401 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     5461 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     5521 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     5581 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     5641 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     5701 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     5761 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     5821 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     5881 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     5941 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     6001 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     6061 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     6121 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     6181 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     6241 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     6301 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     6361 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     6421 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg
     6481 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     6541 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     6601 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     6661 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     6721 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     6781 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     6841 caacgcgcgg ggagaggc
//
