LOCUS       sequence-309517-        5874 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pRSF-Duet1 derived plasmid containing C-terminally hexa-histidine 
            tagged dCas-Phi-1(D371A) 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 5874)
  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 5874)
  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: pPP378
FEATURES             Location/Qualifiers
     source          1..5874
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     897..916
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(936..1013)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        1059..1077
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1078..1102
                     /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             3267..3284
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        3334..3352
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3353..3377
                     /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             3486..3530
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3568..3586)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3582..3629
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3859..4674)
                     /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(3859..4674)
                     /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     4582..4601
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4675..4766)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4782..5531)
                     /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(4792..4809)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(5032..5051)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5727..5874,1..935))
                     /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
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGVSVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
       61 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
      121 cccactaccg agatgtccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
      181 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
      241 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
      301 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
      361 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
      421 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
      481 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
      541 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
      601 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
      661 acacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
      721 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
      781 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
      841 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
      901 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
      961 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     1021 tcgacgctct cccttatgcg actcctgcat taggaaatta atacgactca ctatagggga
     1081 attgtgagcg gataacaatt cccctgtaga aataattttg tttaacttta ataaggagat
     1141 ataccatggc ggacacccca acactgttca cacagttcct ccgacaccat ctaccagggc
     1201 aacgctttcg taaggatatc ctaaagcaag ctggccgaat ccttgccaac aaaggagaag
     1261 acgccacgat tgcgttcttg agaggaaaga gcgaagaatc gccaccagat tttcaaccgc
     1321 ccgtgaagtg tcctatcata gcgtgcagta ggccgctcac ggaatggcca atttatcaag
     1381 cctcggtggc tatacaaggc tacgtttatg gacaaagcct tgccgagttt gaggcttcgg
     1441 atccaggatg tagcaaagat ggtttgcttg ggtggtttga taaaaccgga gtctgcactg
     1501 actacttctc cgtccaaggt ctaaacctga tcttccagaa tgctcgcaag cgctatatcg
     1561 gagtccagac gaaagtcaca aatcgcaacg aaaagcgcca caaaaagctc aagcggatca
     1621 acgctaagcg tatagccgaa ggattgcctg aactaacttc tgatgagcca gagtcagcct
     1681 tagatgaaac tgggcacttg attgatccac ccggcctaaa cactaacatc tactgctacc
     1741 aacaggtaag tcctaaacct ttagcactct ccgaagtgaa tcaattgcca acggcttacg
     1801 cagggtactc cacgagtgga gacgatccca tacaaccgat ggttacgaaa gatcgtctta
     1861 gtatttcaaa gggccagcca gggtatatcc cggagcacca gagagcatta ctttcccaga
     1921 aaaaacatcg ccgtatgcgt ggctatggcc tcaaggccag agcactgcta gtgatcgtaa
     1981 gaatccaaga tgactgggct gtaatcgact tacgttcgtt gctgcggaac gcctactggc
     2041 gtcgtattgt gcagacaaaa gagccaagta caatcacgaa gttactcaaa ctagtgactg
     2101 gggatcctgt actagacgcc acaaggatgg ttgcaacttt cacatataaa ccaggcatag
     2161 ttcaggtgcg ttcagcgaag tgcctcaaga acaagcaagg ttctaagctt ttctcggagc
     2221 gctacctcaa tgagacggta agcgttacta gcatcgccct agggagcaat aacctggttg
     2281 cggttgcgac ttatcgtctt gtcaacggca acacccctga gttgcttcaa cggttcacct
     2341 taccaagtca tttagtaaag gatttcgagc ggtacaagca agctcatgac acactagaag
     2401 actccatcca gaaaaccgcc gtagcgagct tgccgcaagg tcagcaaaca gaaataagaa
     2461 tgtggtccat gtacgggttt agagaggcac aggaacgtgt ttgccaagaa ctcggattag
     2521 ctgatggctc gatcccatgg aatgtcatga cagctacgtc aacgattctc acggatctat
     2581 ttctggcacg tggcggagat ccgaaaaagt gtatgttcac ttctgagcct aagaagaaaa
     2641 aaaactccaa acaggtcttg tacaagattc gagacagagc ctgggccaaa atgtatcgga
     2701 ctcttctgtc gaaagagacg cgagaagctt ggaacaaggc actgtggggg ctcaagagag
     2761 gctctcctga ttatgcacgc ttgtccaaac gtaaagaaga gctggctcgt cgctgcgtca
     2821 actatacgat tagcacggct gagaagagag cccaatgcgg tcggaccatc gttgctctgg
     2881 aggatctcaa cattggtttc ttccatggta gagggaaaca agaacctggg tgggttggat
     2941 tattcacgcg gaagaaggaa aatcgatggc tcatgcaggc tttgcataaa gcgttcttag
     3001 agcttgcaca ccatcgtggc taccacgtta tagaggtcaa tccagcctac acgtcacaaa
     3061 cgtgtccggt gtgtaggcat tgtgacccag acaaccgtga ccagcataat cgagaagctt
     3121 tccactgcat aggatgcggg ttccgtggta atgctgatct cgatgtggcg acccacaaca
     3181 tcgcgatggt tgcaattacc ggagaatcgc tcaagcgtgc gagaggctcg gtggcgtcaa
     3241 aaaccccgca gcctctcgcg gctgaacatc accatcacca tcactaagtc gaacagaaag
     3301 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
     3361 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
     3421 tggcagatct caattggata tcggccggcc acgcgatcgc tgacgtcggt accctcgagt
     3481 ctggtaaaga aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg
     3541 cagcttaatt aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     3601 cctctaaacg ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg
     3661 tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt
     3721 aacgaccctg ccctgaaccg acgacaagct gacgaccggg tcagcgcaag tggcactttt
     3781 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3841 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     3901 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     3961 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     4021 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     4081 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     4141 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     4201 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cggtcgctgt taaaaggaca
     4261 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     4321 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     4381 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     4441 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     4501 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     4561 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     4621 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcatactctt
     4681 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4741 tgaatgtatt tagaaaaata aacaaatagg catgcagcgc tcttccgctt cctcgctcac
     4801 tgactcgcta cgctcggtcg ttcgactgcg gcgagcggtg tcagctcact caaaagcggt
     4861 aatacggtta tccacagaat caggggataa agccggaaag aacatgtgag caaaaagcaa
     4921 agcaccggaa gaagccaacg ccgcaggcgt ttttccatag gctccgcccc cctgacgagc
     4981 atcacaaaaa tcgacgctca agccagaggt ggcgaaaccc gacaggacta taaagatacc
     5041 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5101 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgtt
     5161 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5221 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5281 acgacttatc gccactggca gcagccattg gtaactgatt tagaggactt tgtcttgaag
     5341 ttatgcacct gttaaggcta aactgaaaga acagattttg gtgagtgcgg tcctccaacc
     5401 cacttacctt ggttcaaaga gttggtagct cagcgaacct tgagaaaacc accgttggta
     5461 gcggtggttt ttctttattt atgagatgat gaatcaatcg gtctatcaag tcaacgaaca
     5521 gctattccgt tactctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
     5581 agccccatac gatataagtt gtaattctca tgttagtcat gccccgcgcc caccggaagg
     5641 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     5701 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5761 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     5821 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt cacc
//
