LOCUS       sequence-309574-        7180 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing Homo sapiens codon optimized Cas-Phi-2 and a 
            SapI-GG stuffer spacer for editing in human cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7180)
  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 7180)
  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: pPP441
FEATURES             Location/Qualifiers
     source          1..7180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2281..2301
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2308..2328
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2347..2394
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             2404..2460
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2461..3057
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     2954..2974
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3091..3108)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3097..3304
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3313..3453
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3313..3442
                     /label=AAV2 ITR
     rep_origin      3528..3983
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3615..3634)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3825..3846
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4000..4019)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4119..4141
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4179..4197)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4265..4369
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4370..4438,4439..5230)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4588..4607)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5401..5989
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5890..5909
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        6051..6299
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6051..6071
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6222..6241
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        6371..6656
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        6658..6935
                     /label=chicken beta-actin promoter
     intron          6936..7163
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
ORIGIN
        1 atgccaaagc cagccgtgga gtctgagttt tctaaggtac tcaagaagca ctttccgggc
       61 gagcgattta ggtctagcta catgaagcgg ggtggtaaaa tcttggcagc ccagggtgaa
      121 gaagcggtcg tcgcgtatct gcaaggcaag tccgaggagg aacccccgaa ttttcagccg
      181 ccggcgaaat gtcatgttgt tacgaaatca cgagatttcg ccgagtggcc aattatgaag
      241 gcctccgaag caatccaaag gtatatctat gcgctctcta cgacggaacg ggcagcttgc
      301 aagcctggca aatcttcaga gtcccacgcg gcctggttcg cggcaactgg cgtgtcaaac
      361 cacggttata gccatgttca aggcctcaat cttatcttcg accacacgct gggaagatac
      421 gatggtgttc tgaaaaaggt gcagctgaga aatgagaaag cccgcgcccg gctggaaagt
      481 atcaacgcct ctcgagccga cgaaggactt ccagaaataa aggcagagga ggaagaggtc
      541 gctacaaatg aaaccggaca ccttttgcag cctccgggga tcaacccaag tttctacgtt
      601 taccagacta tttctccgca ggcttacagg ccgcgagatg agattgtact gccgcccgag
      661 tatgccggct acgtccgaga tccgaacgcc cctatccccc ttggcgtggt tcggaatcgg
      721 tgcgatattc agaagggatg ccctggatac atccccgaat ggcaaagaga ggcaggtact
      781 gcaatttccc ctaagacggg taaagccgtc accgttcccg gcctcagtcc aaaaaaaaat
      841 aaacgaatgc gacgatactg gaggtccgag aaagagaagg cccaagatgc actgctcgtt
      901 actgtgagaa tcggcactga ctgggtcgta atcgacgttc gaggtttgct gcggaatgcg
      961 cggtggcgca ccattgcgcc caaggatata tccttgaatg ccctcttgga tctctttaca
     1021 ggcgacccgg tcatagatgt tcggagaaac attgtgactt tcacctacac tctggacgct
     1081 tgcggtacat atgctcgcaa atggactctc aaagggaaac agactaaggc aaccctcgat
     1141 aagttgaccg caacccagac cgtggccctg gtagcaatag accttggaca aaccaatccc
     1201 ataagtgcgg gtatcagtag ggtcacgcaa gaaaacgggg cacttcaatg tgaacctctg
     1261 gatcggttca ctctccctga tgatctgctc aaggatatct ccgcgtaccg aatcgcttgg
     1321 gatcgcaacg aggaggaact gagggctagg tccgtcgaag cgctcccaga agctcaacaa
     1381 gctgaagtga gggctctgga cggcgtttct aaagaaaccg ccaggaccca gctctgcgcg
     1441 gacttcggcc ttgatcccaa acggctgcct tgggataaaa tgagcagcaa caccactttc
     1501 atcagtgaag cgttgcttag taattctgtg tctagagatc aggttttttt tactcctgcg
     1561 cctaaaaagg gagcaaagaa aaaagccccc gttgaagtta tgcggaagga taggacctgg
     1621 gcgagggcct ataaaccacg gctcagtgtg gaagcccaaa agctgaaaaa tgaggccttg
     1681 tgggctctca agcgcacttc tccagaatac ctcaagctga gtcggagaaa agaggagctt
     1741 tgtaggcgaa gtattaacta cgtcattgaa aaaacaagac ggaggacaca atgtcagatc
     1801 gtgatacctg tcatagagga cttgaatgtg cgattctttc acggttcagg gaagcgcctg
     1861 cctggctggg ataatttttt cactgcgaag aaggagaaca ggtggtttat acagggcctc
     1921 cacaaagcat tcagcgactt gcgaactcat cgctccttct acgtattcga agtccgcccg
     1981 gagcggactt caataacgtg cccaaaatgc gggcactgcg aggttgggaa ccgggatggg
     2041 gaggcttttc agtgccttag ttgcggcaaa acgtgcaatg ccgaccttga cgtggctacc
     2101 cataatctga ctcaagtcgc ccttacagga aaaacaatgc cgaaacgcga ggaacctaga
     2161 gatgcccagg gcacagctcc agcccgaaaa acaaagaagg cgtcaaagag caaggctccg
     2221 ccagccgaac gagaggacca aactccagca caggaaccgt cccagacttc cggaagcgga
     2281 cccaagaaaa aacgcaaggt ggaagatcct aagaaaaagc ggaaagtgag cctgggcagc
     2341 ggctccgatt acaaagatga cgatgacaaa gactacaagg atgatgatga taagggatcc
     2401 ggcgcaacaa acttctctct gctgaaacaa gccggagatg tcgaagagaa tcctggaccg
     2461 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     2521 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     2581 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     2641 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     2701 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     2761 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     2821 gcgtggttcc tggccaccgt cggagtctcg cccgaccacc agggcaaggg tctgggcagc
     2881 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     2941 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     3001 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgaacg
     3061 cgttaagaat tcctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat
     3121 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     3181 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     3241 ggggtggggt ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg
     3301 gggagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     3361 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
     3421 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta
     3481 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
     3541 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3601 cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3661 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3721 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     3781 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3841 aactggaaca acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc
     3901 gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     3961 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
     4021 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4081 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     4141 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     4201 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     4261 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4321 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     4381 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     4441 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     4501 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     4561 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     4621 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     4681 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     4741 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     4801 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     4861 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     4921 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     4981 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     5041 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat
     5101 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     5161 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     5221 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     5281 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     5341 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     5401 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     5461 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5521 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     5581 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     5641 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     5701 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     5761 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     5821 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     5881 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     5941 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     6001 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt gagggcctat
     6061 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
     6121 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
     6181 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
     6241 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
     6301 gtcggaacgc tcaacgattg cccctcacga ggggacagaa gagctaatgc tcttcatttt
     6361 ttttggtacc cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     6421 cccgcccatt gacgtcaata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     6481 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     6541 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag tacatgacct
     6601 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg
     6661 aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
     6721 tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggc
     6781 gcgcgccrgg gggggggggg gggggsgggg ggsggggsgg ggcgaggcgg agaggtgcgg
     6841 cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc
     6901 ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc
     6961 ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac
     7021 tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag ctgagcaaga
     7081 ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac ctggagcacc
     7141 tgcctgaaat cacttttttt caggttggac cggtgccacc
//
