LOCUS       sequence-339969-        6600 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of codon-optimized His-EiCsm6 with a TEV 
            protease cleavage site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6600)
  AUTHORS   Liu TY, Knott GJ, Smock DCJ, Desmarais JJ, Son S, Bhuiya A, 
            Jakhanwal S, Prywes N, Agrawal S, Diaz de Leon Derby M, Switz NA, 
            Armstrong M, Harris AR, Charles EJ, Thornton BW, Fozouni P, Shu J, 
            Stephens SI, Kumar GR, Zhao C, Mok A, Iavarone AT, Escajeda AM, 
            McIntosh R, Kim S, Dugan EJ, Pollard KS, Tan MX, Ott M, Fletcher DA,
            Lareau LF, Hsu PD, Savage DF, Doudna JA
  TITLE     Accelerated RNA detection using tandem CRISPR nucleases.
  JOURNAL   Nat Chem Biol. 2021 Sep;17(9):982-988. doi: 
            10.1038/s41589-021-00842-2. Epub 2021 Aug 5.
  PUBMED    34354262
REFERENCE   2  (bases 1 to 6600)
  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"; doi: 
            "10.1038/s41589-021-00842-2"; journalName: "Nat Chem Biol"; date: 
            "2021-09"; volume: "17"; issue: "9"; pages: "982-988"
COMMENT     Sequence Label: pET28a_His-TEV-EiCsm6
FEATURES             Location/Qualifiers
     source          1..6600
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..147
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1465..1482
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1535..1553)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1549..1596
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1633..2088
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1720..1739)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1930..1951
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2180..2995)
                     /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(2180..2995)
                     /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     2903..2922
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2987..3006
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3117..3705
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3606..3625
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3859..3876
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3891..4030
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3956..3975)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4116..4138
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4132..4323)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5132..6214)
                     /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     6176..6195
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6215..6292)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6498..6517
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcgaga atctttattt tcagggccat atgaaaattc tattttcacc cataggaaat
      181 acggacccgt ggcgtaacga ccgcgacggc gcaatgctgc acattgttcg tcactatcag
      241 ccagacagag tcgtgctgtt tttcaccgag agcatctggc agggtaatca gcattttagc
      301 ggccagcaag cgtttgattg ggttaagatt atccaaagta tcaacgagaa ctgccaaatc
      361 gaaatcaagt gcgatactat tgaagttgag aacgactttg acgcatataa ggacctgttc
      421 caccagtact tggtggagga aaaacgtaaa tacccgaatg cggaaatttt cctgaatgtt
      481 acctctggta caccgcagat ggaaaccacc ctgtgtctgg aatacgtaac ctatcccgac
      541 aagatgcgtt gcattcaggt tagcaccccg ctgaaaacga gcaatgctaa aaccaaatat
      601 gctcaggctg attgtcaaga ggttgatttg gagattgtta atgaggaaga gagccagcag
      661 ccgagccgtt gccataaaat cgccatcctg agcttccgcg aagccatcgt ccgtaaccag
      721 attaagtccc tgctggacaa ttatgattat gaagccgcgt tacaactggt tgcgtcgcaa
      781 aagagcttcc gtaacggcaa agagatccgc aagaagctga aagagttgat tgatgacatc
      841 aaaatgcatc gtgttttcag ctatcttatc aagcagtacc cgcgtaacga gaagctgcag
      901 aaagcgctgt tgcacaccat tttgttagaa atgcgtcacc aacgtggtga tatcgcggaa
      961 actctgatcc gtgtgaaatc catcgcggaa tatatcgtgg agcagtacat tcaaaaaaac
     1021 tacccgtacc tgatcattta taaagaagat aagccgtact tcaacgtgtc ctacagccaa
     1081 gagctgaccg aatcttactt ggcgctgatg gattcacgca acaagaagac gaacaagaag
     1141 atgaccgtcg atagcctgga ccgtattctc ggttttccgg cataccgcga cttcctgcaa
     1201 ctgctcgagg cgtcgaacga aatgacgaac gagatgaata aggtcaatga gattaacaac
     1261 ctgcgtaata aagttgcaca taatttagac tctctgaatc tcgaccgcga taaaaacggc
     1321 cgtaagatca ccaacgctgt gaccgcagtg cgcaccatgc tgttggcggt gttcccggaa
     1381 gtgcaggaga acgacttcca ctaccttaaa caatttaacc aatccataaa ggagttgctc
     1441 taaaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac
     1501 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     1561 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     1621 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     1681 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     1741 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     1801 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     1861 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1921 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1981 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     2041 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     2101 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     2161 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     2221 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     2281 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     2341 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     2401 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     2461 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     2521 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     2581 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     2641 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     2701 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     2761 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     2821 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     2881 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     2941 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     3001 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     3061 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     3121 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3181 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     3241 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     3301 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3361 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3421 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3481 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3541 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3601 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3661 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3721 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3781 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3841 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     3901 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     3961 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     4021 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4081 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4141 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     4201 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     4261 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     4321 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     4381 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     4441 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     4501 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     4561 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     4621 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     4681 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     4741 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     4801 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     4861 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     4921 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     4981 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     5041 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     5101 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     5161 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     5221 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     5281 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     5341 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     5401 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     5461 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     5521 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     5581 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     5641 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     5701 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     5761 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     5821 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     5881 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     5941 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     6001 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     6061 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     6121 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     6181 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     6241 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     6301 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     6361 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     6421 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     6481 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     6541 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
//
