LOCUS       sequence-309510-        6024 bp    DNA     circular SYN 14-APR-2025
DEFINITION  pRSF-Duet1 derived plasmid containing C-terminally 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 6024)
  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 6024)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: pPP085
FEATURES             Location/Qualifiers
     source          1..6024
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     549..568
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(588..665)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        711..729
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    730..754
                     /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             3069..3086
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        3136..3154
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3155..3179
                     /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             3288..3332
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3370..3388)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3384..3431
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3661..4476)
                     /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(3661..4476)
                     /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     4384..4403
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4477..4568)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4584..5333)
                     /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(4594..4611)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4834..4853)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5529..6024,1..587))
                     /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 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
       61 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
      121 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
      181 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
      241 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
      301 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
      361 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
      421 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
      481 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
      541 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
      601 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
      661 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaaat taatacgact
      721 cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt tgtttaactt
      781 taataaggag atataccatg ccaaaacccg cggttgagag tgaattctcg aaagtgctta
      841 agaagcactt ccccggggag cgcttccgtt ccagttacat gaaacgcggt ggcaagatct
      901 tagccgccca gggagaagag gccgtagttg catacttaca gggaaaatca gaggaggaac
      961 cgcctaactt ccaacctccc gcgaaatgtc atgtggttac taaaagtcgc gattttgccg
     1021 aatggccgat tatgaaagcc agcgaggcta ttcaacgcta catctacgcg ttgtcaacca
     1081 ctgagcgtgc cgcgtgtaaa ccagggaaaa gcagcgaatc acatgctgcc tggtttgcag
     1141 cgactggcgt cagtaaccac gggtattcac atgtccaagg gcttaactta atctttgacc
     1201 atactctggg gcgttacgat ggggttttga agaaggttca actgcgtaat gaaaaggcac
     1261 gtgcccgttt ggagtcaatt aatgcatctc gtgccgatga gggtttacct gaaatcaagg
     1321 cggaagagga agaggtagcc acaaatgaga cagggcattt gcttcaaccg ccaggcatta
     1381 atcccagctt ctatgtttat caaacgatct cacctcaggc ttatcgtcca cgcgacgaga
     1441 ttgttttgcc ccccgagtat gcagggtacg tgcgtgatcc caacgccccc attccgttag
     1501 gagtagtccg caaccgttgt gatattcaga aaggctgtcc gggctatatt cctgagtggc
     1561 agcgcgaggc aggtactgcg atctctccga agactgggaa agccgttaca gttcctggcc
     1621 tttcccctaa gaagaacaag cgtatgcgcc gctattggcg ttcggaaaaa gagaaggcac
     1681 aagatgcact gttggttacc gtccgtatcg gtactgactg ggtagtgatt gatgtgcgcg
     1741 gattattgcg taatgcccgc tggcgcacca ttgctccaaa ggacatctca ttaaacgcgc
     1801 ttttagacct gttcactgga gacccagtga ttgatgtccg ccgcaacatt gtaactttta
     1861 cctacacgct tgatgcctgc ggtacctatg ctcgtaaatg gacgttaaaa ggaaagcaga
     1921 caaaggcaac actggataaa ctgactgcca cccaaacagt agcgctggta gcgatcgatt
     1981 tgggccagac gaaccctatt tcagcgggaa tctcccgtgt tacgcaggaa aacggggcgc
     2041 ttcaatgtga gccgctggac cgctttacgt tacctgacga cttgttgaaa gatatcagtg
     2101 cgtaccgcat cgcatgggac cgcaacgagg aggaattacg tgcccgttca gtcgaagcgt
     2161 tacccgaagc acagcaggcc gaagtacgtg ctctggacgg cgtaagcaag gagaccgctc
     2221 gtactcaatt atgtgctgat ttcggccttg atcccaagcg tctgccttgg gacaagatgt
     2281 cctcaaacac tacattcatc agcgaagccc tgttgtcaaa tagtgtatcg cgtgaccagg
     2341 ttttctttac cccagctcct aaaaaaggag ccaaaaaaaa agctccggtg gaggttatgc
     2401 gcaaggaccg tacttgggcc cgtgcttata aaccacgtct gtctgttgaa gcccagaaac
     2461 tgaaaaacga agctctgtgg gcccttaaac gtacgagtcc cgaatacctt aaattaagcc
     2521 gtcgtaaaga ggagctttgc cgtcgcagta tcaattacgt tatcgagaag acccgccgtc
     2581 gtacccagtg tcaaatcgtg atcccggtga ttgaagatct gaacgtccgc tttttccacg
     2641 gatctgggaa acgtctgcct ggctgggata atttctttac agcgaaaaaa gagaaccgtt
     2701 ggttcatcca gggccttcac aaggcttttt ccgacttacg cacacatcgc agtttctacg
     2761 ttttcgaggt acgtccagag cgcacctcca ttacctgccc caaatgcggg cactgcgagg
     2821 ttgggaaccg tgatggcgaa gcgttccaat gcctttcctg tggaaaaaca tgtaatgcag
     2881 acttagacgt agccactcac aacctgacac aggttgccct taccggaaaa accatgccta
     2941 aacgtgaaga accacgcgat gcccagggga ccgctccggc acgtaagacc aaaaaagcat
     3001 caaagtcgaa ggccccccct gctgaacgcg aggaccagac cccggcccaa gaacctagcc
     3061 agaccagcca tcaccatcac catcactaag tcgaacagaa agtaatcgta ttgtacacgg
     3121 ccgcataatc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
     3181 catcttagta tattagttaa gtataagaag gagatataca tatggcagat ctcaattgga
     3241 tatcggccgg ccacgcgatc gctgacgtcg gtaccctcga gtctggtaaa gaaaccgctg
     3301 ctgcgaaatt tgaacgccag cacatggact cgtctactag cgcagcttaa ttaacctagg
     3361 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     3421 ggggtttttt gctgaaacct caggcatttg agaagcacac ggtcacactg cttccggtag
     3481 tcaataaacc ggtaaaccag caatagacat aagcggctat ttaacgaccc tgccctgaac
     3541 cgacgacaag ctgacgaccg ggtctccgca agtggcactt ttcggggaaa tgtgcgcgga
     3601 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
     3661 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     3721 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     3781 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     3841 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     3901 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     3961 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     4021 cgcctgagcg agacgaaata cgcggtcgct gttaaaagga caattacaaa caggaatcga
     4081 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     4141 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     4201 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     4261 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     4321 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     4381 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     4441 cctagagcaa gacgtttccc gttgaatatg gctcatactc ttcctttttc aatattattg
     4501 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4561 taaacaaata ggcatgcagc gctcttccgc ttcctcgctc actgactcgc tacgctcggt
     4621 cgttcgactg cggcgagcgg tgtcagctca ctcaaaagcg gtaatacggt tatccacaga
     4681 atcaggggat aaagccggaa agaacatgtg agcaaaaagc aaagcaccgg aagaagccaa
     4741 cgccgcaggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4801 caagccagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4861 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4921 tcccttcggg aagcgtggcg ctttctcata gctcacgctg ttggtatctc agttcggtgt
     4981 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5041 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5101 cagcagccat tggtaactga tttagaggac tttgtcttga agttatgcac ctgttaaggc
     5161 taaactgaaa gaacagattt tggtgagtgc ggtcctccaa cccacttacc ttggttcaaa
     5221 gagttggtag ctcagcgaac cttgagaaaa ccaccgttgg tagcggtggt ttttctttat
     5281 ttatgagatg atgaatcaat cggtctatca agtcaacgaa cagctattcc gttactctag
     5341 atttcagtgc aatttatctc ttcaaatgta gcacctgaag tcagccccat acgatataag
     5401 ttgtaattct catgttagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     5461 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     5521 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5581 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     5641 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     5701 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     5761 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca
     5821 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     5881 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     5941 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     6001 tatttatgcc agccagccag acgc
//
