LOCUS       sequence-306501-        7030 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing Homo sapiens codon optimized Cas-Phi-1 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 7030)
  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 7030)
  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: pPP394
FEATURES             Location/Qualifiers
     source          1..7030
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2133..2153
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2160..2180
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2199..2246
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             2256..2312
                     /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             2313..2909
                     /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     2806..2826
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2943..2960)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2949..3156
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3165..3305
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3165..3294
                     /label=AAV2 ITR
     rep_origin      3380..3835
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3467..3486)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3677..3698
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3852..3871)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3971..3993
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4031..4049)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4117..4221
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4222..4290,4291..5082)
                     /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(4440..4459)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5253..5841
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5742..5761
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        5903..6151
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5903..5923
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6074..6093
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        6223..6508
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        6510..6787
                     /label=chicken beta-actin promoter
     intron          6788..7015
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      join(7027..7030,1..6)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 ccatggcgga cacaccaaca cttttcacac agtttcttcg gcatcatctg cccggccagc
       61 ggttccgaaa agacatattg aagcaagccg gacgaatact cgccaacaag ggtgaggacg
      121 caacgatagc tttcctcagg ggaaagtcag aagagtctcc gcctgatttt cagcctccgg
      181 tgaagtgccc cataatcgcg tgctcccggc cactcacgga atggccaatc tatcaagcat
      241 ctgtggctat ccagggatat gtgtatgggc aaagcctcgc agagtttgaa gcgtccgatc
      301 ctggttgctc taaagatggc ttgttggggt ggttcgacaa aaccggtgta tgtacggatt
      361 atttttctgt acaagggctt aacctcatat ttcagaacgc gagaaaaaga tatattgggg
      421 tgcaaacaaa ggtaactaat cgcaacgaga aaagacataa aaagctcaag aggataaacg
      481 cgaaaaggat agcggagggt ctccctgaat tgacctccga cgagccggag agcgcgctgg
      541 atgagaccgg tcatcttatt gacccaccag gacttaatac caatatctat tgttaccaac
      601 aagtttcccc gaaaccgctt gcattgtctg aagtgaacca attgccaacg gcttatgcgg
      661 ggtattcaac gtccggtgac gaccctatcc agccgatggt aactaaagat aggctgagca
      721 taagtaaagg ccaacccggg tacattcccg agcaccaacg ggctctgctg tctcaaaaga
      781 aacatagacg gatgagagga tacggcctta aagcaagggc gctgttggtt attgtgagaa
      841 tccaagatga ctgggccgtg atcgatctcc gatccttgct gaggaacgcg tactggcggc
      901 gcatcgtgca aacaaaagaa ccgtctacta ttacaaagct ccttaaactg gtaactggtg
      961 accccgtttt ggatgcaacc agaatggtag cgacatttac ttacaaacca ggtatagtgc
     1021 aggtccgaag cgccaagtgt ctgaagaaca agcaagggag taagttgttt tctgaacgct
     1081 atttgaacga aactgtgagt gtaaccagta ttgaccttgg gtcaaataac ctcgttgctg
     1141 tagcgacgta ccgcctggtt aatggcaata caccggagct cctccaaagg ttcacacttc
     1201 caagtcacct ggtaaaagat ttcgagcgct acaagcaagc acatgatact ttggaagatt
     1261 ctattcaaaa gactgctgtg gcaagcctgc ctcaaggcca acaaacggaa atacgcatgt
     1321 ggtctatgta cgggttcaga gaggctcaag aacgcgtctg tcaggagctg ggtttggctg
     1381 atggaagcat cccttggaac gtgatgaccg cgacgagcac gattctgacc gatttgttcc
     1441 tcgcaagggg aggcgatcct aagaagtgta tgtttacctc cgagccaaag aagaaaaaga
     1501 acagcaaaca ggtactttat aaaatcaggg accgggcctg ggccaaaatg tatcgaacac
     1561 ttctttcaaa ggagacgcgc gaagcatgga ataaagcatt gtggggactt aagcggggca
     1621 gcccggatta cgctaggctt agtaagagga aagaagagtt ggccagaagg tgtgtaaatt
     1681 acacgatatc tactgctgaa aaacgcgcgc agtgtggccg cacaatcgtg gctttggagg
     1741 accttaatat cggcttcttt cacggaaggg gtaagcagga acccggatgg gtcgggctgt
     1801 tcaccaggaa gaaggaaaac cggtggctga tgcaagcctt gcataaggcc tttctcgagt
     1861 tggcgcacca tcgaggttat catgttatag aggttaatcc tgcgtatact tcccaaacat
     1921 gtcctgtatg caggcattgc gatccggata acagggacca gcataaccga gaagcattcc
     1981 actgtatagg atgcggattc cgcggtaatg cggacttgga cgtagccacc cacaacatag
     2041 cgatggtagc cataacggga gagtcattga agcgagcacg gggctcagtt gctagtaaaa
     2101 ctcctcaacc cttggccgca gagggaagcg gacccaagaa aaaacgcaag gtggaagatc
     2161 ctaagaaaaa gcggaaagtg agcctgggca gcggctccga ttacaaagat gacgatgaca
     2221 aagactacaa ggatgatgat gataagggat ccggcgcaac aaacttctct ctgctgaaac
     2281 aagccggaga tgtcgaagag aatcctggac cgaccgagta caagcccacg gtgcgcctcg
     2341 ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc
     2401 ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag
     2461 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
     2521 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
     2581 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
     2641 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggagtct
     2701 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
     2761 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
     2821 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
     2881 ggtgcatgac ccgcaagccc ggtgcctgaa cgcgttaaga attcctagag ctcgctgatc
     2941 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     3001 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     3061 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     3121 ggaggattgg gaagagaata gcaggcatgc tggggagcgg ccgcaggaac ccctagtgat
     3181 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     3241 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     3301 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     3361 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     3421 gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc
     3481 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     3541 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     3601 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     3661 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg
     3721 ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag
     3781 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     3841 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     3901 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     3961 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     4021 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     4081 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     4141 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     4201 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     4261 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     4321 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     4381 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     4441 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     4501 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     4561 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     4621 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     4681 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     4741 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     4801 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     4861 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     4921 ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga tggtaagccc
     4981 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     5041 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     5101 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     5161 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     5221 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     5281 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     5341 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     5401 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     5461 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     5521 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     5581 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     5641 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     5701 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     5761 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     5821 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     5881 tgctggcctt ttgctcacat gtgagggcct atttcccatg attccttcat atttgcatat
     5941 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
     6001 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
     6061 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
     6121 tttatatatc ttgtggaaag gacgaaacac cgggagagat ctcaaacgat tgctcgatta
     6181 gtcgagacag aagagctaat gctcttcatt ttttttggta cccgttacat aacttacggt
     6241 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc
     6301 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     6361 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     6421 gcccgcctgg cattgtgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     6481 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct
     6541 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
     6601 tgcagcgatg ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg ggsggggsgr
     6661 ggggsggggs ggggsgrggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg
     6721 aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg
     6781 gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg
     6841 ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc
     6901 ttctcctccg ggctgtaatt agctgagcaa gaggtaaggg tttaagggat ggttggttgg
     6961 tggggtatta atgtttaatt acctggagca cctgcctgaa atcacttttt ttcaggttgg
     7021 accggtgcca
//
