LOCUS       sequence-309576-        5874 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pRSF-Duet1 derived plasmid containing C-terminally hexa-histidine 
            tagged Cas-Phi-1 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: pPP076
FEATURES             Location/Qualifiers
     source          1..5874
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     525..544
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(564..641)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        687..705
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    706..730
                     /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             2895..2912
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        2962..2980
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2981..3005
                     /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             3114..3158
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3196..3214)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3210..3257
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3487..4302)
                     /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(3487..4302)
                     /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     4210..4229
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4303..4394)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4410..5159)
                     /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(4420..4437)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4660..4679)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5355..5874,1..563))
                     /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 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
       61 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
      121 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
      181 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
      241 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctgac acccagttga
      301 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
      361 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
      421 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
      481 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
      541 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
      601 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
      661 cttatgcgac tcctgcatta ggaaattaat acgactcact ataggggaat tgtgagcgga
      721 taacaattcc cctgtagaaa taattttgtt taactttaat aaggagatat accatggcgg
      781 acaccccaac actgttcaca cagttcctcc gacaccatct accagggcaa cgctttcgta
      841 aggatatcct aaagcaagct ggccgaatcc ttgccaacaa aggagaagac gccacgattg
      901 cgttcttgag aggaaagagc gaagaatcgc caccagattt tcaaccgccc gtgaagtgtc
      961 ctatcatagc gtgcagtagg ccgctcacgg aatggccaat ttatcaagcc tcggtggcta
     1021 tacaaggcta cgtttatgga caaagccttg ccgagtttga ggcttcggat ccaggatgta
     1081 gcaaagatgg tttgcttggg tggtttgata aaaccggagt ctgcactgac tacttctccg
     1141 tccaaggtct aaacctgatc ttccagaatg ctcgcaagcg ctatatcgga gtccagacga
     1201 aagtcacaaa tcgcaacgaa aagcgccaca aaaagctcaa gcggatcaac gctaagcgta
     1261 tagccgaagg attgcctgaa ctaacttctg atgagccaga gtcagcctta gatgaaactg
     1321 ggcacttgat tgatccaccc ggcctaaaca ctaacatcta ctgctaccaa caggtaagtc
     1381 ctaaaccttt agcactctcc gaagtgaatc aattgccaac ggcttacgca gggtactcca
     1441 cgagtggaga cgatcccata caaccgatgg ttacgaaaga tcgtcttagt atttcaaagg
     1501 gccagccagg gtatatcccg gagcaccaga gagcattact ttcccagaaa aaacatcgcc
     1561 gtatgcgtgg ctatggcctc aaggccagag cactgctagt gatcgtaaga atccaagatg
     1621 actgggctgt aatcgactta cgttcgttgc tgcggaacgc ctactggcgt cgtattgtgc
     1681 agacaaaaga gccaagtaca atcacgaagt tactcaaact agtgactggg gatcctgtac
     1741 tagacgccac aaggatggtt gcaactttca catataaacc aggcatagtt caggtgcgtt
     1801 cagcgaagtg cctcaagaac aagcaaggtt ctaagctttt ctcggagcgc tacctcaatg
     1861 agacggtaag cgttactagc atcgatctag ggagcaataa cctggttgcg gttgcgactt
     1921 atcgtcttgt caacggcaac acccctgagt tgcttcaacg gttcacctta ccaagtcatt
     1981 tagtaaagga tttcgagcgg tacaagcaag ctcatgacac actagaagac tccatccaga
     2041 aaaccgccgt agcgagcttg ccgcaaggtc agcaaacaga aataagaatg tggtccatgt
     2101 acgggtttag agaggcacag gaacgtgttt gccaagaact cggattagct gatggctcga
     2161 tcccatggaa tgtcatgaca gctacgtcaa cgattctcac ggatctattt ctggcacgtg
     2221 gcggagatcc gaaaaagtgt atgttcactt ctgagcctaa gaagaaaaaa aactccaaac
     2281 aggtcttgta caagattcga gacagagcct gggccaaaat gtatcggact cttctgtcga
     2341 aagagacgcg agaagcttgg aacaaggcac tgtgggggct caagagaggc tctcctgatt
     2401 atgcacgctt gtccaaacgt aaagaagagc tggctcgtcg ctgcgtcaac tatacgatta
     2461 gcacggctga gaagagagcc caatgcggtc ggaccatcgt tgctctggag gatctcaaca
     2521 ttggtttctt ccatggtaga gggaaacaag aacctgggtg ggttggatta ttcacgcgga
     2581 agaaggaaaa tcgatggctc atgcaggctt tgcataaagc gttcttagag cttgcacacc
     2641 atcgtggcta ccacgttata gaggtcaatc cagcctacac gtcacaaacg tgtccggtgt
     2701 gtaggcattg tgacccagac aaccgtgacc agcataatcg agaagctttc cactgcatag
     2761 gatgcgggtt ccgtggtaat gctgatctcg atgtggcgac ccacaacatc gcgatggttg
     2821 caattaccgg agaatcgctc aagcgtgcga gaggctcggt ggcgtcaaaa accccgcagc
     2881 ctctcgcggc tgaacatcac catcaccatc actaagtcga acagaaagta atcgtattgt
     2941 acacggccgc ataatcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
     3001 attccccatc ttagtatatt agttaagtat aagaaggaga tatacatatg gcagatctca
     3061 attggatatc ggccggccac gcgatcgctg acgtcggtac cctcgagtct ggtaaagaaa
     3121 ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc tactagcgca gcttaattaa
     3181 cctaggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     3241 tcttgagggg ttttttgctg aaacctcagg catttgagaa gcacacggtc acactgcttc
     3301 cggtagtcaa taaaccggta aaccagcaat agacataagc ggctatttaa cgaccctgcc
     3361 ctgaaccgac gacaagctga cgaccgggtc tccgcaagtg gcacttttcg gggaaatgtg
     3421 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     3481 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     3541 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     3601 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     3661 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     3721 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     3781 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     3841 tgattgcgcc tgagcgagac gaaatacgcg gtcgctgtta aaaggacaat tacaaacagg
     3901 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     3961 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     4021 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     4081 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     4141 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     4201 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     4261 tcgcggccta gagcaagacg tttcccgttg aatatggctc atactcttcc tttttcaata
     4321 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     4381 gaaaaataaa caaataggca tgcagcgctc ttccgcttcc tcgctcactg actcgctacg
     4441 ctcggtcgtt cgactgcggc gagcggtgtc agctcactca aaagcggtaa tacggttatc
     4501 cacagaatca ggggataaag ccggaaagaa catgtgagca aaaagcaaag caccggaaga
     4561 agccaacgcc gcaggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4621 gacgctcaag ccagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4681 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4741 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgttgg tatctcagtt
     4801 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4861 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4921 cactggcagc agccattggt aactgattta gaggactttg tcttgaagtt atgcacctgt
     4981 taaggctaaa ctgaaagaac agattttggt gagtgcggtc ctccaaccca cttaccttgg
     5041 ttcaaagagt tggtagctca gcgaaccttg agaaaaccac cgttggtagc ggtggttttt
     5101 ctttatttat gagatgatga atcaatcggt ctatcaagtc aacgaacagc tattccgtta
     5161 ctctagattt cagtgcaatt tatctcttca aatgtagcac ctgaagtcag ccccatacga
     5221 tataagttgt aattctcatg ttagtcatgc cccgcgccca ccggaaggag ctgactgggt
     5281 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     5341 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5401 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     5461 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     5521 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     5581 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc
     5641 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     5701 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     5761 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     5821 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taat
//
