LOCUS       sequence-396471-        4512 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of HA-tagged CASP sigma in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4512)
  AUTHORS   Strecker J, Demircioglu FE, Li D, Faure G, Wilkinson ME, Gootenberg 
            JS, Abudayyeh OO, Nishimasu H, Macrae RK, Zhang F
  TITLE     RNA-activated protein cleavage with a CRISPR-associated 
            endopeptidase.
  JOURNAL   Science. 2022 Nov 25;378(6622):874-881. doi: 
            10.1126/science.add7450. Epub 2022 Nov 3.
  PUBMED    36423276
REFERENCE   2  (bases 1 to 4512)
  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: 
            "10.1126/science.add7450"; journalName: "Science"; date: 
            "2022-11-25- 25"; volume: "378"; issue: "6622"; pages: "874-881"
COMMENT     Sequence Label: pCDF_HA_CASP_sigma
FEATURES             Location/Qualifiers
     source          1..4512
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     CDS             74..100
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     RBS             754..762
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     promoter        880..898
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    899..923
                     /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)."
     CDS             1032..1076
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1114..1132)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1128..1175
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1346..2137)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(2138..2229)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(3290..4372)
                     /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     4334..4353
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4373..4450)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(4496..4512,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgtacccat acgatgttcc agattacgcc ggagccacca tgtcattaaa
      121 tcacccctgt ctaagtgctc gtgccgctag cccggaatgc accgtttgtc cggtttttaa
      181 agccggtcat caggaggcgt tcgtcaagat cttggaaaag aaggtgcgtt ttttggagtc
      241 ccgcaaacac ttgtcgccag aggacaaaga ggatatcctg agcgaatttg tcctgcagat
      301 tcagagctcc acctcctctt ttaagggcca gaatgcaaaa ttcgagacgt gggcgtatcg
      361 tatcttccaa aacgtgtggg ctgatttttt ccgcaaaatc aagaacttta agacccaacg
      421 tactgacgac ggcatttaca tccaagaaac gattaaagtg aaaaagaccg acaaggacgg
      481 ccgtgaaatg ctggaggaaa aagcgcgtac gggtgttttc ctgccgtata aagagaacat
      541 tgaggaccgc atcgaaagca gagagatctt cagcattttc cgcaaggcgg ttaaagatcc
      601 gaaaaaggcg ttctgctacc acctgctggt taatcattac acctggaaaa gcgaaggtct
      661 gaaacaagat gaaatggcag cgcgttatga aatgcgtcac gatacctttc gtaagcggct
      721 gtctgagtgc cgtagccgct tcaagcagta ccttaaggag gtgacccagt aagatccgaa
      781 ttcgagctcg gcgcgcctgc aggtcgacaa gcttgcggcc gcataatgct taagtcgaac
      841 agaaagtaat cgtattgtac acggccgcat aatcgaaatt aatacgactc actatagggg
      901 aattgtgagc ggataacaat tccccatctt agtatattag ttaagtataa gaaggagata
      961 tacatatggc agatctcaat tggatatcgg ccggccacgc gatcgctgac gtcggtaccc
     1021 tcgagtctgg taaagaaacc gctgctgcga aatttgaacg ccagcacatg gactcgtcta
     1081 ctagcgcagc ttaattaacc taggctgctg ccaccgctga gcaataacta gcataacccc
     1141 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa acctcaggca tttgagaagc
     1201 acacggtcac actgcttccg gtagtcaata aaccggtaaa ccagcaatag acataagcgg
     1261 ctatttaacg accctgccct gaaccgacga ccgggtcatc gtggccggat cttgcggccc
     1321 ctcggcttga acgaattgtt agacattatt tgccgactac cttggtgatc tcgcctttca
     1381 cgtagtggac aaattcttcc aactgatctg cgcgcgaggc caagcgatct tcttcttgtc
     1441 caagataagc ctgtctagct tcaagtatga cgggctgata ctgggccggc aggcgctcca
     1501 ttgcccagtc ggcagcgaca tccttcggcg cgattttgcc ggttactgcg ctgtaccaaa
     1561 tgcgggacaa cgtaagcact acatttcgct catcgccagc ccagtcgggc ggcgagttcc
     1621 atagcgttaa ggtttcattt agcgcctcaa atagatcctg ttcaggaacc ggatcaaaga
     1681 gttcctccgc cgctggacct accaaggcaa cgctatgttc tcttgctttt gtcagcaaga
     1741 tagccagatc aatgtcgatc gtggctggct cgaagatacc tgcaagaatg tcattgcgct
     1801 gccattctcc aaattgcagt tcgcgcttag ctggataacg ccacggaatg atgtcgtcgt
     1861 gcacaacaat ggtgacttct acagcgcgga gaatctcgct ctctccaggg gaagccgaag
     1921 tttccaaaag gtcgttgatc aaagctcgcc gcgttgtttc atcaagcctt acggtcaccg
     1981 taaccagcaa atcaatatca ctgtgtggct tcaggccgcc atccactgcg gagccgtaca
     2041 aatgtacggc cagcaacgtc ggttcgagat ggcgctcgat gacgccaact acctctgata
     2101 gttgagtcga tacttcggcg atcaccgctt ccctcatact cttccttttt caatattatt
     2161 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     2221 ataaacaaat agctagctca ctcggtcgct acgctccggg cgtgagactg cggcgggcgc
     2281 tgcggacaca tacaaagtta cccacagatt ccgtggataa gcaggggact aacatgtgag
     2341 gcaaaacagc agggccgcgc cggtggcgtt tttccatagg ctccgccctc ctgccagagt
     2401 tcacataaac agacgctttt ccggtgcatc tgtgggagcc gtgaggctca accatgaatc
     2461 tgacagtacg ggcgaaaccc gacaggactt aaagatcccc accgtttccg gcgggtcgct
     2521 ccctcttgcg ctctcctgtt ccgaccctgc cgtttaccgg atacctgttc cgcctttctc
     2581 ccttacggga agtgtggcgc tttctcatag ctcacacact ggtatctcgg ctcggtgtag
     2641 gtcgttcgct ccaagctggg ctgtaagcaa gaactccccg ttcagcccga ctgctgcgcc
     2701 ttatccggta actgttcact tgagtccaac ccggaaaagc acggtaaaac gccactggca
     2761 gcagccattg gtaactggga gttcgcagag gatttgttta gctaaacacg cggttgctct
     2821 tgaagtgtgc gccaaagtcc ggctacactg gaaggacaga tttggttgct gtgctctgcg
     2881 aaagccagtt accacggtta agcagttccc caactgactt aaccttcgat caaaccacct
     2941 ccccaggtgg ttttttcgaa ggttaagtca gttggggaac tgcttaacct tcgatcaaac
     3001 cacctcccca ggtggttttt tcgtttacag ggcaaaagat tacgcgcaga aaaaaaggat
     3061 ctcaagaaga tcctttgatc ttttctactg aaccgctcta gatttcagtg caatttatct
     3121 cttcaaatgt agcacctgaa gtcagcccca tacgatataa gttgtaattc tcatgttagt
     3181 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     3241 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     3301 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3361 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     3421 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     3481 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     3541 cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg
     3601 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     3661 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     3721 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     3781 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     3841 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     3901 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     3961 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     4021 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     4081 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     4141 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     4201 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     4261 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     4321 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     4381 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     4441 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaaattaata
     4501 cgactcacta ta
//
