LOCUS       sequence-309569-        6051 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pRSF-Duet1 derived plasmid containing C-terminally hexa-histidine 
            tagged dCas-Phi-3(D413A) in MCS1 for expression in E. coli and 
            protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6051)
  AUTHORS   Pausch P, Al-Shayeb B, Bisom-Rapp E, Tsuchida CA, Li Z, Cress BF, 
            Knott GJ, Jacobsen SE, Banfield JF, Doudna JA
  TITLE     CRISPR-CasPhi from huge phages is a hypercompact genome editor.
  JOURNAL   Science. 2020 Jul 17;369(6501):333-337. doi: 
            10.1126/science.abb1400.
  PUBMED    32675376
REFERENCE   2  (bases 1 to 6051)
  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"; 
            journalName: "Science"; date: "2020-07-17- 17"; volume: "369"; 
            issue: "6501"; pages: "333-337"
COMMENT     Sequence Label: pPP384
FEATURES             Location/Qualifiers
     source          1..6051
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     294..313
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(333..410)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        456..474
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    475..499
                     /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             2841..2858
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        2908..2926
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2927..2951
                     /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             3060..3104
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3142..3160)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3156..3203
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3433..4248)
                     /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(3433..4248)
                     /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     4156..4175
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4249..4340)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4356..5105)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(4366..4383)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4606..4625)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5301..6051,1..332))
                     /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 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
       61 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
      121 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
      181 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
      241 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
      301 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
      361 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
      421 acgctctccc ttatgcgact cctgcattag gaaattaata cgactcacta taggggaatt
      481 gtgagcggat aacaattccc ctgtagaaat aattttgttt aactttaata aggagatata
      541 ccatggaaaa agagattact gagctaacta aaatacgaag agagtttcca aataagaaat
      601 tttcatctac ggatatgaaa aaagcaggta aacttctaaa ggcagaagga cctgatgctg
      661 tgcgcgattt tcttaatagc tgtcaagaaa taataggtga ttttaaacct ccagttaaaa
      721 ctaatattgt ttctataagt aggccatttg aagagtggcc agttagtatg gttggaagag
      781 ctattcaaga atattatttt agtttgacta aggaagagtt ggagtcagtt catccaggaa
      841 ctagtagtga agaccataaa tcatttttta atataactgg attgagcaat tacaattata
      901 caagcgtaca gggattgaat ttaatattca agaatgcaaa ggctatttat gatggcacat
      961 tagttaaggc aaataataaa aataagaaat tagaaaaaaa attcaacgaa attaatcata
     1021 aacgttcatt agagggtctg cctattatca ctccagattt tgaagagcca tttgatgaga
     1081 atggtcattt gaataatcct cctggaataa atcgaaatat atatggttat caaggttgtg
     1141 cggcaaaggt ttttgttcca tctaaacaca agatggtttc tttaccaaaa gaatatgagg
     1201 ggtataatag ggatcctaat ttatcattag ctgggtttcg taatcgtttg gaaattcctg
     1261 aaggtgaacc tggtcatgtg ccatggtttc agagaatgga tattccagag gggcagattg
     1321 gacatgtgaa caaaattcag cgttttaatt ttgtacatgg aaagaactct ggtaaggtaa
     1381 agttttcaga caaaacaggt agagtaaaaa gatatcatca ttcaaaatat aaagacgcta
     1441 ccaaaccata taagttttta gaagagtcaa aaaaagtctc tgcgttagat tctattctcg
     1501 ctatcataac tattggtgat gattgggtag tttttgatat tcgtggtttg tatagaaatg
     1561 ttttttatag agagcttgcc caaaaaggtt taacggctgt ccaattgtta gatttgttta
     1621 ctggagatcc tgtaattgat cctaaaaagg gcgttgttac attttcatat aaagagggtg
     1681 ttgttcctgt attttcacaa aagattgtac ctcgctttaa atctagggat acattagaaa
     1741 aacttacttc acaaggacct gtagctttat tatctgtagc gcttggacaa aatgaacctg
     1801 tagcagctcg tgtatgttca ttaaaaaaca taaatgataa gataacttta gataactctt
     1861 gcagaatttc atttttagat gattataaaa aacaaattaa agattatcgt gacagcttag
     1921 atgaattaga gataaaaatt agattagagg ctataaattc actagagaca aatcagcagg
     1981 ttgagattcg tgatctggat gtgttttctg cagatcgtgc aaaagcaaat acagttgata
     2041 tgtttgatat agatccaaat ctaatttcat gggatagtat gtcagacgcc cgtgtttcaa
     2101 cacagatatc agatttgtat ctaaaaaatg gaggtgatga gtctagagta tattttgaaa
     2161 ttaataataa gcgtataaaa cgatcagatt ataatatttc acaactagtt agaccaaaat
     2221 tatctgatag tactagaaaa aatctaaatg attctatttg gaaattgaag agaactagtg
     2281 aagagtatct taaactatct aaaagaaagt tagaattatc tagagcggtt gttaattata
     2341 caattcgaca atctaaattg ttgagtggaa ttaatgatat tgtaattatt cttgaagatt
     2401 tagatgtcaa gaaaaaattt aacggacgtg ggataagaga tattggttgg gataattttt
     2461 tcagttcaag aaaggaaaat cgttggttta ttccagcatt tcataaagct tttagtgaat
     2521 tgagtagtaa tcgtggattg tgtgttattg aggttaatcc tgcttggaca agtgctacgt
     2581 gccctgattg tggattttgt agcaaagaaa atagagatgg tattaatttt acttgtcgga
     2641 agtgtggtgt tagctatcat gcagatattg atgtagccac gctgaatatt gctcgggtgg
     2701 ctgttttggg aaagccaatg tctggacccg ctgatcgcga gaggttaggt gatacgaaaa
     2761 aaccaagggt cgctcgcagt cgtaaaacta tgaaaagaaa ggatatttca aacagtacgg
     2821 tggaagctat ggttacggcc catcaccatc accatcacta agtcgaacag aaagtaatcg
     2881 tattgtacac ggccgcataa tcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     2941 ataacaattc cccatcttag tatattagtt aagtataaga aggagatata catatggcag
     3001 atctcaattg gatatcggcc ggccacgcga tcgctgacgt cggtaccctc gagtctggta
     3061 aagaaaccgc tgctgcgaaa tttgaacgcc agcacatgga ctcgtctact agcgcagctt
     3121 aattaaccta ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     3181 aacgggtctt gaggggtttt ttgctgaaac ctcaggcatt tgagaagcac acggtcacac
     3241 tgcttccggt agtcaataaa ccggtaaacc agcaatagac ataagcggct atttaacgac
     3301 cctgccctga accgacgaca agctgacgac cgggtctccg caagtggcac ttttcgggga
     3361 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3421 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     3481 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     3541 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     3601 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     3661 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     3721 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     3781 cattcgtgat tgcgcctgag cgagacgaaa tacgcggtcg ctgttaaaag gacaattaca
     3841 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     3901 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     3961 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     4021 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     4081 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     4141 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     4201 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcatac tcttcctttt
     4261 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     4321 tatttagaaa aataaacaaa taggcatgca gcgctcttcc gcttcctcgc tcactgactc
     4381 gctacgctcg gtcgttcgac tgcggcgagc ggtgtcagct cactcaaaag cggtaatacg
     4441 gttatccaca gaatcagggg ataaagccgg aaagaacatg tgagcaaaaa gcaaagcacc
     4501 ggaagaagcc aacgccgcag gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4561 aaaatcgacg ctcaagccag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4621 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4681 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgttggtatc
     4741 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4801 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4861 tatcgccact ggcagcagcc attggtaact gatttagagg actttgtctt gaagttatgc
     4921 acctgttaag gctaaactga aagaacagat tttggtgagt gcggtcctcc aacccactta
     4981 ccttggttca aagagttggt agctcagcga accttgagaa aaccaccgtt ggtagcggtg
     5041 gtttttcttt atttatgaga tgatgaatca atcggtctat caagtcaacg aacagctatt
     5101 ccgttactct agatttcagt gcaatttatc tcttcaaatg tagcacctga agtcagcccc
     5161 atacgatata agttgtaatt ctcatgttag tcatgccccg cgcccaccgg aaggagctga
     5221 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     5281 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5341 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     5401 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     5461 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     5521 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     5581 atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     5641 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     5701 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     5761 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     5821 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5881 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     5941 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     6001 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg c
//
