LOCUS       sequence-339968-        7057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of codon-optimized His-SUMO-TtCsm6 with a TEV 
            protease cleavage site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7057)
  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 7057)
  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: pET_His6-SUMO-TEV-TtCsm6
FEATURES             Location/Qualifiers
     source          1..7057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     973..992
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1012..1089)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1295..1314
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1398..1417
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1398..1416
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1417..1441
                     /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             1456..1478
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1497..1514
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1533..1826
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             1833..1853
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3318..3335
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3388..3406)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3402..3449
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3486..3941
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3573..3592)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3783..3804
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4034..4849)
                     /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(4034..4849)
                     /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     4757..4776
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4841..4860
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4971..5559
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5460..5479
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5713..5730
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5745..5884
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5810..5829)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5970..5992
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5986..6177)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6986..7057,1..1011))
                     /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 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
       61 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
      121 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
      181 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
      241 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
      301 gataccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
      361 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
      421 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
      481 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
      541 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
      601 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
      661 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
      721 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
      781 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
      841 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
      901 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
      961 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     1021 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     1081 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     1141 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     1201 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     1261 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     1321 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     1381 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     1441 ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggt tcttctcacc
     1501 atcaccatca ccatggttct tctatggcta gcatgtcgga ctcagaagtc aatcaagaag
     1561 ctaagccaga ggtcaagcca gaagtcaagc ctgagactca catcaattta aaggtgtccg
     1621 atggatcttc agagatcttc ttcaagatca aaaagaccac tcctttaaga aggctgatgg
     1681 aagcgttcgc taaaagacag ggtaaggaaa tggactcctt aagattcttg tacgacggta
     1741 ttagaattca agctgatcag acccctgaag atttggacat ggaggataac gatattattg
     1801 aggctcacag agaacagatt ggtgggatcg aggaaaacct gtacttccaa tccaatgcag
     1861 aggacctgga tgcgctgtgg gaacgctatc gtgaggcggt tcgtgcgggt ggtaatccgc
     1921 aggcgctgta tcaagagatg gtgtggccgg cgctgctggc gctgtggcgt gaaaagccgc
     1981 gtgtttaccc gtttccgcag gcgttcgctg tgagcgttca caccctgggt accagcccgg
     2041 aggcgaccgc gctggcgatc ctgggtgcgg gcgcggagcg tgtgtatgtt ctgcacaccc
     2101 cggaaagcgc gcgttttctg ccgcgtctgc gtcaagacac cggcaaggat ctgtacccgg
     2161 tggaaatcgg taaaagcgac gttgaggcga tttatcgtga agtgaagcgt ctgctggaga
     2221 aacacccgga agtgccggtt gcgctggatc tgaccagcgg taccaaagcg atgagcgcgg
     2281 gtctggcggc ggcgggtttc tttttccagc gtttttaccc gaaagtgcgt gtggtttacg
     2341 ttgacaacga ggactatgat ccggaactgc gtcgtccgcg tgcgggtacc gagaaactgc
     2401 gtattctgcc gaacccgcac gaggcgctgg cggaagttga tgcgctgttc gcgaaggagc
     2461 tgtacggtaa aggcgaattt ggtcaggcgg cggcgtattt ccgtggtatg gtgggccgta
     2521 ccggtaacca agcgtacgcg ctgtatgcgc tgctggcgga gatgtaccgt gcgtggcgtg
     2581 cgctggactt cggtgaagcg ctgaaggcgg gccgtaaact gctgggtcag ctgagccaaa
     2641 acgtttggct gaaccacccg ctgaacgcgc gtcgtgaggc gctggaagcg caagtggcgc
     2701 tgctggaggc ggttgaccgt tttctgaagg cgcgtgattt cgcgctgaaa gaaggcgttt
     2761 atggtctggc gcgtaccctg ctgcacctgg cgcaggaagc gaaggaagag gcggcggtgc
     2821 tggcggcgct gtatgcgtat cgtgcgctgg agctgctgct gcaagaacgt ctggcgctgc
     2881 tgggtcgtcg tgcggaagcg ccgggtctga gcccggagga agcggaggcg ctgcgtaaag
     2941 cgctggcgga actgctgggc gtgctgccgg aggaagttcg tctgccggcg aagctgggtc
     3001 tgctggacct gctggcgttt ctgcgtctga aaggcgatga ggcgctgggt cgtctgagcc
     3061 tggcggaact gcgtggcctg gcgggtgcgc tgaaaggtcg taacagcgcg ctgctggtgc
     3121 acggtttcga tgttccgagc ccgaaagcgg tggagggtat tgcgcgtctg gcgcagggtc
     3181 tgctgcaaga tctggaggcg cgtaccgcgc tgggtccgct gagcccggag ccggtgccgc
     3241 tgggttttta ataacattgg aagtggataa cggatccgaa ttcgagcgcc gtcgacaagc
     3301 ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
     3361 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     3421 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     3481 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     3541 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3601 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3661 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     3721 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     3781 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     3841 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     3901 atttaacgcg aattttaaca aactagtaac gtttacaatt tcaggtggca cttttcgggg
     3961 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     4021 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     4081 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     4141 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     4201 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     4261 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
     4321 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     4381 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
     4441 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atgttttcac
     4501 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     4561 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     4621 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     4681 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     4741 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     4801 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     4861 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     4921 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4981 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     5041 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     5101 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     5161 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     5221 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     5281 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     5341 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     5401 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     5461 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     5521 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     5581 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     5641 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     5701 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     5761 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     5821 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     5881 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     5941 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     6001 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     6061 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     6121 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     6181 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     6241 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     6301 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     6361 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     6421 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     6481 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     6541 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     6601 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     6661 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     6721 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     6781 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     6841 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     6901 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     6961 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     7021 ccagctgcat taatgaatcg gccaacgcgc ggggaga
//
