LOCUS       sequence-309579-        5820 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pRSF-Duet1 derived plasmid containing C-terminally 2xSV40 NLS and 
            hexa-histidine tagged Cas-Phi-2 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 5820)
  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 5820)
  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: pPP537
FEATURES             Location/Qualifiers
     source          1..5820
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     947..966
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(986..1063)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1109..1128
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1109..1127
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1128..1152
                     /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             3476..3496
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3503..3523
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3536..3553
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3564..3582)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3578..3625
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3855..4670)
                     /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(3855..4670)
                     /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     4578..4597
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4671..4762)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4778..5527)
                     /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(4788..4805)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(5028..5047)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5723..5820,1..985))
                     /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 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
       61 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
      121 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
      181 agatgtccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
      241 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
      301 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
      361 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
      421 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
      481 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
      541 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
      601 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
      661 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
      721 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
      781 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
      841 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
      901 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
      961 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     1021 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     1081 cccttatgcg actcctgcat taggaaatta atacgactca ctatagggga attgtgagcg
     1141 gataacaatt cccctgtaga aataattttg tttaacttta ataaggagat ataccatgcc
     1201 aaaacccgcg gttgagagtg aattctcgaa agtgcttaag aagcacttcc ccggggagcg
     1261 cttccgttcc agttacatga aacgcggtgg caagatctta gccgcccagg gagaagaggc
     1321 cgtagttgca tacttacagg gaaaatcaga ggaggaaccg cctaacttcc aacctcccgc
     1381 gaaatgtcat gtggttacta aaagtcgcga ttttgccgaa tggccgatta tgaaagccag
     1441 cgaggctatt caacgctaca tctacgcgtt gtcaaccact gagcgtgccg cgtgtaaacc
     1501 agggaaaagc agcgaatcac atgctgcctg gtttgcagcg actggcgtca gtaaccacgg
     1561 gtattcacat gtccaagggc ttaacttaat ctttgaccat actctggggc gttacgatgg
     1621 ggttttgaag aaggttcaac tgcgtaatga aaaggcacgt gcccgtttgg agtcaattaa
     1681 tgcatctcgt gccgatgagg gtttacctga aatcaaggcg gaagaggaag aggtagccac
     1741 aaatgagaca gggcatttgc ttcaaccgcc aggcattaat cccagcttct atgtttatca
     1801 aacgatctca cctcaggctt atcgtccacg cgacgagatt gttttgcccc ccgagtatgc
     1861 agggtacgtg cgtgatccca acgcccccat tccgttagga gtagtccgca accgttgtga
     1921 tattcagaaa ggctgtccgg gctatattcc tgagtggcag cgcgaggcag gtactgcgat
     1981 ctctccgaag actgggaaag ccgttacagt tcctggcctt tcccctaaga agaacaagcg
     2041 tatgcgccgc tattggcgtt cggaaaaaga gaaggcacaa gatgcactgt tggttaccgt
     2101 ccgtatcggt actgactggg tagtgattga tgtgcgcgga ttattgcgta atgcccgctg
     2161 gcgcaccatt gctccaaagg acatctcatt aaacgcgctt ttagacctgt tcactggaga
     2221 cccagtgatt gatgtccgcc gcaacattgt aacttttacc tacacgcttg atgcctgcgg
     2281 tacctatgct cgtaaatgga cgttaaaagg aaagcagaca aaggcaacac tggataaact
     2341 gactgccacc caaacagtag cgctggtagc gatcgatttg ggccagacga accctatttc
     2401 agcgggaatc tcccgtgtta cgcaggaaaa cggggcgctt caatgtgagc cgctggaccg
     2461 ctttacgtta cctgacgact tgttgaaaga tatcagtgcg taccgcatcg catgggaccg
     2521 caacgaggag gaattacgtg cccgttcagt cgaagcgtta cccgaagcac agcaggccga
     2581 agtacgtgct ctggacggcg taagcaagga gaccgctcgt actcaattat gtgctgattt
     2641 cggccttgat cccaagcgtc tgccttggga caagatgtcc tcaaacacta cattcatcag
     2701 cgaagccctg ttgtcaaata gtgtatcgcg tgaccaggtt ttctttaccc cagctcctaa
     2761 aaaaggagcc aaaaaaaaag ctccggtgga ggttatgcgc aaggaccgta cttgggcccg
     2821 tgcttataaa ccacgtctgt ctgttgaagc ccagaaactg aaaaacgaag ctctgtgggc
     2881 ccttaaacgt acgagtcccg aataccttaa attaagccgt cgtaaagagg agctttgccg
     2941 tcgcagtatc aattacgtta tcgagaagac ccgccgtcgt acccagtgtc aaatcgtgat
     3001 cccggtgatt gaagatctga acgtccgctt tttccacgga tctgggaaac gtctgcctgg
     3061 ctgggataat ttctttacag cgaaaaaaga gaaccgttgg ttcatccagg gccttcacaa
     3121 ggctttttcc gacttacgca cacatcgcag tttctacgtt ttcgaggtac gtccagagcg
     3181 cacctccatt acctgcccca aatgcgggca ctgcgaggtt gggaaccgtg atggcgaagc
     3241 gttccaatgc ctttcctgtg gaaaaacatg taatgcagac ttagacgtag ccactcacaa
     3301 cctgacacag gttgccctta ccggaaaaac catgcctaaa cgtgaagaac cacgcgatgc
     3361 ccaggggacc gctccggcac gtaagaccaa aaaagcatca aagtcgaagg ccccccctgc
     3421 tgaacgcgag gaccagaccc cggcccaaga acctagccag accagcgggt cagggcctaa
     3481 gaagaagcgt aaagtagaag atcctaagaa aaaacgcaag gtgggcagca gcggacacca
     3541 ccatcatcac cattaagctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     3601 taaacgggtc ttgaggggtt ttttgctgaa acctcaggca tttgagaagc acacggtcac
     3661 actgcttccg gtagtcaata aaccggtaaa ccagcaatag acataagcgg ctatttaacg
     3721 accctgccct gaaccgacga caagctgacg accgggtctc cgcaagtggc acttttcggg
     3781 gaaatgtgtg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     3841 tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
     3901 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
     3961 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
     4021 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca
     4081 ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact
     4141 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
     4201 ttcattcgtg attgcgcctg agcgagacga aatacgcggt cgctgttaaa aggacaatta
     4261 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     4321 cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg
     4381 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     4441 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
     4501 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca
     4561 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     4621 gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat actcttcctt
     4681 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     4741 tgtatttaga aaaataaaca aataggcatg cagcgctctt ccgcttcctc gctcactgac
     4801 tcgctacgct cggtcgttcg actgcggcga gcggtgtcag ctcactcaaa agcggtaata
     4861 cggttatcca cagaatcagg ggataaagcc ggaaagaaca tgtgagcaaa aagcaaagca
     4921 ccggaagaag ccaacgccgc aggcgttttt ccataggctc cgcccccctg acgagcatca
     4981 caaaaatcga cgctcaagcc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5041 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5101 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgttggta
     5161 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5221 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5281 cttatcgcca ctggcagcag ccattggtaa ctgatttaga ggactttgtc ttgaagttat
     5341 gcacctgtta aggctaaact gaaagaacag attttggtga gtgcggtcct ccaacccact
     5401 taccttggtt caaagagttg gtagctcagc gaaccttgag aaaaccaccg ttggtagcgg
     5461 tggtttttct ttatttatga gatgatgaat caatcggtct atcaagtcaa cgaacagcta
     5521 ttccgttact ctagatttca gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc
     5581 ccatacgata taagttgtaa ttctcatgtt agtcatgccc cgcgcccacc ggaaggagct
     5641 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     5701 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5761 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
//
