LOCUS       sequence-309523-        6024 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pRSF-Duet1 derived plasmid containing C-terminally hexa-histidine 
            tagged dCas-Phi-2(D394A) 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 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: pPP381
FEATURES             Location/Qualifiers
     source          1..6024
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     810..829
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(849..926)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        972..990
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    991..1015
                     /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             3330..3347
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        3397..3415
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3549..3593
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3631..3649)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3645..3692
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3922..4737)
                     /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(3922..4737)
                     /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     4645..4664
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4738..4829)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4845..5594)
                     /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(4855..4872)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(5095..5114)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5790..6024,1..848))
                     /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 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg
       61 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
      121 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
      181 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
      241 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
      301 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
      361 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
      421 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
      481 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
      541 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
      601 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
      661 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
      721 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
      781 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
      841 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
      901 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
      961 cattaggaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctgt
     1021 agaaataatt ttgtttaact ttaataagga gatataccat gccaaaaccc gcggttgaga
     1081 gtgaattctc gaaagtgctt aagaagcact tccccgggga gcgcttccgt tccagttaca
     1141 tgaaacgcgg tggcaagatc ttagccgccc agggagaaga ggccgtagtt gcatacttac
     1201 agggaaaatc agaggaggaa ccgcctaact tccaacctcc cgcgaaatgt catgtggtta
     1261 ctaaaagtcg cgattttgcc gaatggccga ttatgaaagc cagcgaggct attcaacgct
     1321 acatctacgc gttgtcaacc actgagcgtg ccgcgtgtaa accagggaaa agcagcgaat
     1381 cacatgctgc ctggtttgca gcgactggcg tcagtaacca cgggtattca catgtccaag
     1441 ggcttaactt aatctttgac catactctgg ggcgttacga tggggttttg aagaaggttc
     1501 aactgcgtaa tgaaaaggca cgtgcccgtt tggagtcaat taatgcatct cgtgccgatg
     1561 agggtttacc tgaaatcaag gcggaagagg aagaggtagc cacaaatgag acagggcatt
     1621 tgcttcaacc gccaggcatt aatcccagct tctatgttta tcaaacgatc tcacctcagg
     1681 cttatcgtcc acgcgacgag attgttttgc cccccgagta tgcagggtac gtgcgtgatc
     1741 ccaacgcccc cattccgtta ggagtagtcc gcaaccgttg tgatattcag aaaggctgtc
     1801 cgggctatat tcctgagtgg cagcgcgagg caggtactgc gatctctccg aagactggga
     1861 aagccgttac agttcctggc ctttccccta agaagaacaa gcgtatgcgc cgctattggc
     1921 gttcggaaaa agagaaggca caagatgcac tgttggttac cgtccgtatc ggtactgact
     1981 gggtagtgat tgatgtgcgc ggattattgc gtaatgcccg ctggcgcacc attgctccaa
     2041 aggacatctc attaaacgcg cttttagacc tgttcactgg agatccagtg attgatgtcc
     2101 gccgcaacat tgtaactttt acctacacgc ttgatgcctg cggtacctat gctcgtaaat
     2161 ggacgttaaa aggaaagcag acaaaggcaa cactggataa actgactgcc acccaaacag
     2221 tagcgctggt agcgatcgcc ttgggccaga cgaaccctat ttcagcggga atctcccgtg
     2281 ttacgcagga aaacggggcg cttcaatgtg agccgctgga ccgctttacg ttacctgacg
     2341 acttgttgaa agatatcagt gcgtaccgca tcgcatggga ccgcaacgag gaggaattac
     2401 gtgcccgttc agtcgaagcg ttacccgaag cacagcaggc cgaagtacgt gctctggacg
     2461 gcgtaagcaa ggaaaccgct cgtactcaat tatgtgctga tttcggcctt gatcccaagc
     2521 gtctgccttg ggacaagatg tcctcaaaca ctacattcat cagcgaagcc ctgttgtcaa
     2581 atagtgtatc gcgtgaccag gttttcttta ccccagctcc taaaaaagga gccaaaaaaa
     2641 aagctccggt ggaggttatg cgcaaggacc gtacttgggc ccgtgcttat aaaccacgtc
     2701 tgtctgttga agcccagaaa ctgaaaaacg aagctctgtg ggcccttaaa cgtacgagtc
     2761 ccgaatacct taaattaagc cgtcgtaaag aggagctttg ccgtcgcagt atcaattacg
     2821 ttatcgagaa gacccgccgt cgtacccagt gtcaaatcgt gatcccggtg attgaagatc
     2881 tgaacgtccg ctttttccac ggatctggga aacgtctgcc tggctgggat aatttcttta
     2941 cagcgaaaaa agagaaccgt tggttcatcc agggccttca caaggctttt tccgacttac
     3001 gcacacatcg cagtttctac gttttcgagg tacgtccaga gcgcacctcc attacctgcc
     3061 ccaaatgcgg gcactgcgag gttgggaacc gtgatggcga agcgttccaa tgcctttcct
     3121 gtggaaaaac atgtaatgca gacttagacg tagccactca caacctgaca caggttgccc
     3181 ttaccggaaa aaccatgcct aaacgtgaag aaccacgcga tgcccagggg accgctccgg
     3241 cacgtaagac caaaaaagca tcaaagtcga aggccccccc tgctgaacgc gaggaccaga
     3301 ccccggccca agaacctagc cagaccagcc atcaccatca ccatcactaa gtcgaacaga
     3361 aagtaatcgt attgtacacg gccgcataat cgaaattaat acgactcact ataggggaat
     3421 tgtgagagga taacaattcc ccatcttagt atattagtta agtataagaa ggagatatac
     3481 atatggcaga tctcaattgg atatcggccg gccacgcgat cgctgacgtc ggtaccctcg
     3541 agtctggtaa agaaaccgct gctgcgaaat ttgaacgcca gcacatggac tcgtctacta
     3601 gcgcagctta attaacctag gctgctgcca ccgctgagca ataactagca taaccccttg
     3661 gggcctctaa acgggtcttg aggggttttt tgctgaaacc tcaggcattt gagaagcaca
     3721 cggtcacact gcttccggta gtcaataaac cggtaaacca gcaatagaca taagcggcta
     3781 tttaacgacc ctgccctgaa ccgacgacaa gctgacgacc gggtctccgc aagtggcact
     3841 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3901 tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     3961 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     4021 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     4081 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     4141 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     4201 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     4261 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcggtcgc tgttaaaagg
     4321 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     4381 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     4441 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     4501 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     4561 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     4621 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     4681 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcatact
     4741 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     4801 atttgaatgt atttagaaaa ataaacaaat aggcatgcag cgctcttccg cttcctcgct
     4861 cactgactcg ctacgctcgg tcgttcgact gcggcgagcg gtgtcagctc actcaaaagc
     4921 ggtaatacgg ttatccacag aatcagggga taaagccgga aagaacatgt gagcaaaaag
     4981 caaagcaccg gaagaagcca acgccgcagg cgtttttcca taggctccgc ccccctgacg
     5041 agcatcacaa aaatcgacgc tcaagccaga ggtggcgaaa cccgacagga ctataaagat
     5101 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5161 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5221 gttggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5281 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5341 gacacgactt atcgccactg gcagcagcca ttggtaactg atttagagga ctttgtcttg
     5401 aagttatgca cctgttaagg ctaaactgaa agaacagatt ttggtgagtg cggtcctcca
     5461 acccacttac cttggttcaa agagttggta gctcagcgaa ccttgagaaa accaccgttg
     5521 gtagcggtgg tttttcttta tttatgagat gatgaatcaa tcggtctatc aagtcaacga
     5581 acagctattc cgttactcta gatttcagtg caatttatct cttcaaatgt agcacctgaa
     5641 gtcagcccca tacgatataa gttgtaattc tcatgttagt catgccccgc gcccaccgga
     5701 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     5761 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5821 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5881 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     5941 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     6001 tgtttgatgg tggttaacgg cggg
//
