LOCUS       sequence-309513-        6051 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pRSF-Duet1 derived plasmid containing C-terminally hexa-histidine 
            tagged Cas-Phi-3 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: pPP089
FEATURES             Location/Qualifiers
     source          1..6051
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(73..1155)
                     /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     1117..1136
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1156..1233)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        1279..1297
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1298..1322
                     /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             3664..3681
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        3731..3749
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3750..3774
                     /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             3883..3927
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3965..3983)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3979..4026
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4256..5071)
                     /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(4256..5071)
                     /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     4979..4998
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5072..5163)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5179..5928)
                     /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(5189..5206)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(5429..5448)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
       61 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
      121 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
      181 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
      241 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
      301 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatgtccgc
      361 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
      421 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
      481 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
      541 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
      601 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
      661 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
      721 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
      781 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
      841 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
      901 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
      961 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     1021 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     1081 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     1141 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     1201 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     1261 actcctgcat taggaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     1321 cccctgtaga aataattttg tttaacttta ataaggagat ataccatgga aaaagagatt
     1381 actgagctaa ctaaaatacg aagagagttt ccaaataaga aattttcatc tacggatatg
     1441 aaaaaagcag gtaaacttct aaaggcagaa ggacctgatg ctgtgcgcga ttttcttaat
     1501 agctgtcaag aaataatagg tgattttaaa cctccagtta aaactaatat tgtttctata
     1561 agtaggccat ttgaagagtg gccagttagt atggttggaa gagctattca agaatattat
     1621 tttagtttga ctaaggaaga gttggagtca gttcatccag gaactagtag tgaagaccat
     1681 aaatcatttt ttaatataac tggattgagc aattacaatt atacaagcgt acagggattg
     1741 aatttaatat tcaagaatgc aaaggctatt tatgatggca cattagttaa ggcaaataat
     1801 aaaaataaga aattagaaaa aaaattcaac gaaattaatc ataaacgttc attagagggt
     1861 ctgcctatta tcactccaga ttttgaagag ccatttgatg agaatggtca tttgaataat
     1921 cctcctggaa taaatcgaaa tatatatggt tatcaaggtt gtgcggcaaa ggtttttgtt
     1981 ccatctaaac acaagatggt ttctttacca aaagaatatg aggggtataa tagggatcct
     2041 aatttatcat tagctgggtt tcgtaatcgt ttggaaattc ctgaaggtga acctggtcat
     2101 gtgccatggt ttcagagaat ggatattcca gaggggcaga ttggacatgt gaacaaaatt
     2161 cagcgtttta attttgtaca tggaaagaac tctggtaagg taaagttttc agacaaaaca
     2221 ggtagagtaa aaagatatca tcattcaaaa tataaagacg ctaccaaacc atataagttt
     2281 ttagaagagt caaaaaaagt ctctgcgtta gattctattc tcgctatcat aactattggt
     2341 gatgattggg tagtttttga tattcgtggt ttgtatagaa atgtttttta tagagagctt
     2401 gcccaaaaag gtttaacggc tgtccaattg ttagatttgt ttactggaga tcctgtaatt
     2461 gatcctaaaa agggcgttgt tacattttca tataaagagg gtgttgttcc tgtattttca
     2521 caaaagattg tacctcgctt taaatctagg gatacattag aaaaacttac ttcacaagga
     2581 cctgtagctt tattatctgt agatcttgga caaaatgaac ctgtagcagc tcgtgtatgt
     2641 tcattaaaaa acataaatga taagataact ttagataact cttgcagaat ttcattttta
     2701 gatgattata aaaaacaaat taaagattat cgtgacagct tagatgaatt agagataaaa
     2761 attagattag aggctataaa ttcactagag acaaatcagc aggttgagat tcgtgatctg
     2821 gatgtgtttt ctgcagatcg tgcaaaagca aatacagttg atatgtttga tatagatcca
     2881 aatctaattt catgggatag tatgtcagac gcccgtgttt caacacagat atcagatttg
     2941 tatctaaaaa atggaggtga tgagtctaga gtatattttg aaattaataa taagcgtata
     3001 aaacgatcag attataatat ttcacaacta gttagaccaa aattatctga tagtactaga
     3061 aaaaatctaa atgattctat ttggaaattg aagagaacta gtgaagagta tcttaaacta
     3121 tctaaaagaa agttagaatt atctagagcg gttgttaatt atacaattcg acaatctaaa
     3181 ttgttgagtg gaattaatga tattgtaatt attcttgaag atttagatgt caagaaaaaa
     3241 tttaacggac gtgggataag agatattggt tgggataatt ttttcagttc aagaaaggaa
     3301 aatcgttggt ttattccagc atttcataaa gcttttagtg aattgagtag taatcgtgga
     3361 ttgtgtgtta ttgaggttaa tcctgcttgg acaagtgcta cgtgccctga ttgtggattt
     3421 tgtagcaaag aaaatagaga tggtattaat tttacttgtc ggaagtgtgg tgttagctat
     3481 catgcagata ttgatgtagc cacgctgaat attgctcggg tggctgtttt gggaaagcca
     3541 atgtctggac ccgctgatcg cgagaggtta ggtgatacga aaaaaccaag ggtcgctcgc
     3601 agtcgtaaaa ctatgaaaag aaaggatatt tcaaacagta cggtggaagc tatggttacg
     3661 gcccatcacc atcaccatca ctaagtcgaa cagaaagtaa tcgtattgta cacggccgca
     3721 taatcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttccccatct
     3781 tagtatatta gttaagtata agaaggagat atacatatgg cagatctcaa ttggatatcg
     3841 gccggccacg cgatcgctga cgtcggtacc ctcgagtctg gtaaagaaac cgctgctgcg
     3901 aaatttgaac gccagcacat ggactcgtct actagcgcag cttaattaac ctaggctgct
     3961 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     4021 tttttgctga aacctcaggc atttgagaag cacacggtca cactgcttcc ggtagtcaat
     4081 aaaccggtaa accagcaata gacataagcg gctatttaac gaccctgccc tgaaccgacg
     4141 acaagctgac gaccgggtct ccgcaagtgg cacttttcgg ggaaatgtgc gcggaacccc
     4201 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
     4261 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     4321 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     4381 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     4441 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     4501 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     4561 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     4621 gagcgagacg aaatacgcgg tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     4681 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     4741 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     4801 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     4861 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     4921 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     4981 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag
     5041 agcaagacgt ttcccgttga atatggctca tactcttcct ttttcaatat tattgaagca
     5101 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5161 aaataggcat gcagcgctct tccgcttcct cgctcactga ctcgctacgc tcggtcgttc
     5221 gactgcggcg agcggtgtca gctcactcaa aagcggtaat acggttatcc acagaatcag
     5281 gggataaagc cggaaagaac atgtgagcaa aaagcaaagc accggaagaa gccaacgccg
     5341 caggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagc
     5401 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     5461 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5521 tcgggaagcg tggcgctttc tcatagctca cgctgttggt atctcagttc ggtgtaggtc
     5581 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5641 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5701 gccattggta actgatttag aggactttgt cttgaagtta tgcacctgtt aaggctaaac
     5761 tgaaagaaca gattttggtg agtgcggtcc tccaacccac ttaccttggt tcaaagagtt
     5821 ggtagctcag cgaaccttga gaaaaccacc gttggtagcg gtggtttttc tttatttatg
     5881 agatgatgaa tcaatcggtc tatcaagtca acgaacagct attccgttac tctagatttc
     5941 agtgcaattt atctcttcaa atgtagcacc tgaagtcagc cccatacgat ataagttgta
     6001 attctcatgt tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt g
//
