LOCUS       sequence-309578-        7207 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing Homo sapiens codon optimized Cas-Phi-3 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 7207)
  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 7207)
  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: pPP444
FEATURES             Location/Qualifiers
     source          1..7207
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        100..385
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        387..664
                     /label=chicken beta-actin promoter
     intron          665..892
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      904..913
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3217..3237
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3244..3264
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3283..3330
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             3340..3396
                     /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             3397..3993
                     /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     3890..3910
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4027..4044)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4033..4240
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4249..4389
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4249..4378
                     /label=AAV2 ITR
     rep_origin      4464..4919
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4551..4570)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4761..4782
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4936..4955)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5055..5077
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5115..5133)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5201..5305
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5306..5374,5375..6166)
                     /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(5524..5543)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6337..6925
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6826..6845
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(6987..7207,1..28)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6987..7007
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7158..7177
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 atatatcttg tggaaaggac gaaacaccga ccaaaacgac tattgattgc ccagtacgct
       61 gggacagaag agctaatgct cttcattttt tttggtaccc gttacataac ttacggtaaa
      121 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaatag taacgccaat
      181 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      241 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      301 cgcctggcat tgtgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      361 cgtattagtc atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc
      421 catctccccc ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc
      481 agcgatgggg gcgggggggg ggggggggcg cgcgccrggg ggggsggggg ggggsgrggg
      541 gsggggsggg gsgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      601 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
      661 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
      721 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
      781 tcctccgggc tgtaattagc tgagcaagag gtaagggttt aagggatggt tggttggtgg
      841 ggtattaatg tttaattacc tggagcacct gcctgaaatc actttttttc aggttggacc
      901 ggtgccacca tggaaaaaga aataactgag ctcaccaaga ttaggcgcga gtttccgaat
      961 aaaaagttca gcagcactga tatgaagaag gcaggtaagt tgttgaaggc agaaggtcct
     1021 gatgctgtta gagacttcct gaactcctgc caggagatta tcggggattt taagccgcct
     1081 gtaaagacaa acatagtcag catatcacga ccctttgagg agtggcctgt tagtatggtg
     1141 gggcgcgcca tccaggaata ttactttagt ttgacaaaag aggaattgga gtccgtccat
     1201 cccggaactt ccagcgagga tcacaagtcc ttctttaaca taactggcct gagcaattac
     1261 aattatacgt cagtccaagg cttgaatctc atcttcaaaa atgcgaaggc catatacgac
     1321 gggactctgg ttaaagcaaa caataaaaat aagaagttgg aaaaaaagtt caatgagatt
     1381 aaccacaagc gaagccttga ggggcttcct ataattacgc cggatttcga ggaacccttt
     1441 gatgagaatg gccatctgaa taatccgcca ggtattaatc gaaatattta cggctaccaa
     1501 ggatgtgccg ctaaagtatt cgttccttcc aagcataaaa tggtatccct ccctaaagaa
     1561 tacgaagggt acaaccggga tccgaacctg tccttggcgg gcttccgaaa tcggctcgag
     1621 ataccggagg gggagcccgg tcacgtgcca tggtttcagc gcatggatat cccggaaggc
     1681 cagatcgggc acgtaaataa gattcaacga ttcaatttcg ttcatggcaa gaattcagga
     1741 aaagtcaaat tcagcgataa gacaggacgg gtaaaacgct accatcattc caagtataaa
     1801 gatgccacta agccttacaa atttcttgaa gaatccaaga aagtcagtgc tctggactcc
     1861 atccttgcca ttatcacaat cggtgatgac tgggtagtgt ttgacattcg cggtctgtat
     1921 agaaatgttt tttatcgcga actggcacag aagggcctga cagcagtgca gctgctggat
     1981 ctgtttacgg gggatccggt gattgacccg aagaagggcg ttgtgacatt cagctataag
     2041 gaaggcgtgg ttccagtatt ttcacagaag atcgttccaa ggttcaagag tcgagacacg
     2101 ctcgagaaat tgaccagtca aggacctgtg gcgctgctct cagtcgacct cggccaaaat
     2161 gaaccagtgg cggcaagggt ttgtagcttg aagaacataa atgataagat cacattggat
     2221 aattcttgca gaatctcctt cctggatgac tacaaaaaac aaatcaaaga ctacagagat
     2281 tccctggacg aacttgaaat caagatacga ctggaagcaa tcaattctct ggaaactaac
     2341 caacaagtag aaattcgcga cctggatgta ttcagtgctg atcgggcaaa ggcaaacact
     2401 gtagatatgt tcgacatcga cccaaatttg atatcctggg attcaatgag cgacgcgagg
     2461 gtgagcacgc aaataagcga tctttatctg aagaatgggg gtgacgaatc tcgagtatat
     2521 ttcgaaatta acaacaaacg gataaagcga tctgattata acattagtca gctggtgagg
     2581 ccaaagcttt ccgacagcac tcggaagaat ctgaacgatt ctatatggaa gttgaaaaga
     2641 actagtgaag aatatttgaa attgtccaaa cgaaagttgg aactgagcag agctgttgtg
     2701 aactacacta tccgccagag caagctcctc tccggaatta acgacattgt tataatactt
     2761 gaggacctgg atgtaaaaaa aaaattcaat ggcaggggca ttcgagatat cggatgggac
     2821 aacttcttca gctccaggaa agagaacagg tggttcattc cggcattcca taaggctttc
     2881 tcagagcttt caagcaaccg gggcctctgt gtcatcgaag tcaacccggc atggacatct
     2941 gccacctgtc ccgactgcgg gttctgtagt aaagagaaca gagatggcat taattttacc
     3001 tgtcgcaagt gcggtgtctc ttaccacgcg gacatagatg ttgccactct taatatagcc
     3061 cgggtggccg ttctcggcaa gcctatgtcc ggacccgccg accgcgagag actgggcgat
     3121 actaagaaac cccgggtagc aaggagccga aagactatga aacggaaaga tattagcaat
     3181 agcaccgttg aggctatggt tacagccgga agcggaccca agaaaaaacg caaggtggaa
     3241 gatcctaaga aaaagcggaa agtgagcctg ggcagcggct ccgattacaa agatgacgat
     3301 gacaaagact acaaggatga tgatgataag ggatccggcg caacaaactt ctctctgctg
     3361 aaacaagccg gagatgtcga agagaatcct ggaccgaccg agtacaagcc cacggtgcgc
     3421 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     3481 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     3541 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     3601 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     3661 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     3721 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcgga
     3781 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     3841 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     3901 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     3961 acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt aagaattcct agagctcgct
     4021 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     4081 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     4141 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     4201 agggggagga ttgggaagag aatagcaggc atgctgggga gcggccgcag gaacccctag
     4261 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     4321 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     4381 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4441 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4501 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc
     4561 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4621 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     4681 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4741 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat
     4801 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa
     4861 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     4921 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     4981 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     5041 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     5101 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     5161 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5221 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5281 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5341 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5401 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5461 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5521 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5581 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5641 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5701 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     5761 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     5821 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     5881 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     5941 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6001 atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa
     6061 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6121 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6181 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6241 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6301 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6361 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6421 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6481 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6541 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6601 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6661 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6721 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6781 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6841 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6901 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     6961 cttttgctgg ccttttgctc acatgtgagg gcctatttcc catgattcct tcatatttgc
     7021 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     7081 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     7141 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     7201 tggcttt
//
