LOCUS       sequence-194593-        7624 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dSa Cas9 fused to optimized combination of truncated 
            activation domains from SCP1 promoter with dual snRP1 poly 
            adenylation signal, and Sa sgRNA for Afp, vector allows for strong 
            activation of mouse Afp, can be packaged and delivered as AAV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7624)
  TITLE     Church Lab CRISPR plasmids 2017
REFERENCE   2  (bases 1 to 7624)
  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"
COMMENT     Sequence Label: pAAV-SCP1-dSa VPR mini.-2X snRP-1 Afp
FEATURES             Location/Qualifiers
     source          1..7624
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(5..27)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     127..146
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(163..618)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(300..321)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     512..531
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   693..833
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   704..833
                     /label=AAV2 ITR
     promoter        842..1090
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     842..862
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1013..1032
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1112..1187
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     regulatory      1294..1303
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1306..1326
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4507..4554
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4585..4734
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             4759..4779
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4939..5295
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     repeat_region   5579..5719
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5579..5708
                     /label=AAV2 ITR
     rep_origin      complement(5781..6369)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5861..5880)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6540..7331,7332..7400))
                     /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     7163..7182
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7401..7505)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7573..7591
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
       61 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
      121 actatgcggc atcagagcag attgtactga gagtgcacca taaaattgta aacgttaata
      181 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caatagaccg
      241 aaatcggcaa aatcccttat aaatcaaaag aatagcccga gatagagttg agtgttgttc
      301 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
      361 ccgtctatca gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttggggt
      421 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
      481 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
      541 aggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
      601 cgccgctaca gggcgcgtac tatggttgct ttgacgtatg cggtgtgaaa taccgcacag
      661 atgcgtaagg agaaaatacc gcatcaggcg cccctgcagg cagctgcgcg ctcgctcgct
      721 cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt
      781 gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt cctgcggccg
      841 cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
      901 gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
      961 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     1021 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     1081 acgaaacacc gctaacttca acgtaaggaa agttttagta ctctggaaac agaatctact
     1141 aaaacaaggc aaaatgccgt gtttatctcg tcaacttgtt ggcgagattt ttttggccgc
     1201 ctcgaggtac ttatataagg gggtgggggc gcgttcgtcc tcagtcgcga tcgaacactc
     1261 gagccgagca gacgtgccta cggaccgacc ggtgccacca tggccccaaa gaagaagcgg
     1321 aaggtcggta tccacggagt cccagcagcc aagcggaact acatcctggg cctggccatc
     1381 ggcatcacca gcgtgggcta cggcatcatc gactacgaga cacgggacgt gatcgatgcc
     1441 ggcgtgcggc tgttcaaaga ggccaacgtg gaaaacaacg agggcaggcg gagcaagaga
     1501 ggcgccagaa ggctgaagcg gcggaggcgg catagaatcc agagagtgaa gaagctgctg
     1561 ttcgactaca acctgctgac cgaccacagc gagctgagcg gcatcaaccc ctacgaggcc
     1621 agagtgaagg gcctgagcca gaagctgagc gaggaagagt tctctgccgc cctgctgcac
     1681 ctggccaaga gaagaggcgt gcacaacgtg aacgaggtgg aagaggacac cggcaacgag
     1741 ctgtccacca aagagcagat cagccggaac agcaaggccc tggaagagaa atacgtggcc
     1801 gaactgcagc tggaacggct gaagaaagac ggcgaagtgc ggggcagcat caacagattc
     1861 aagaccagcg actacgtgaa agaagccaaa cagctgctga aggtgcagaa ggcctaccac
     1921 cagctggacc agagcttcat cgacacctac atcgacctgc tggaaacccg gcggacctac
     1981 tatgagggac ctggcgaggg cagccccttc ggctggaagg acatcaaaga atggtacgag
     2041 atgctgatgg gccactgcac ctacttcccc gaggaactgc ggagcgtgaa gtacgcctac
     2101 aacgccgacc tgtacaacgc cctgaacgac ctgaacaatc tcgtgatcac cagggacgag
     2161 aacgagaagc tggaatatta cgagaagttc cagatcatcg agaacgtgtt caagcagaag
     2221 aagaagccca ccctgaagca gatcgccaaa gaaatcctcg tgaacgaaga ggatattaag
     2281 ggctacagag tgaccagcac cggcaagccc gagttcacca acctgaaggt gtaccacgac
     2341 atcaaggaca ttaccgcccg gaaagagatt attgagaacg ccgagctgct ggatcagatt
     2401 gccaagatcc tgaccatcta ccagagcagc gaggacatcc aggaagaact gaccaatctg
     2461 aactccgagc tgacccagga agagatcgag cagatctcta atctgaaggg ctataccggc
     2521 acccacaacc tgagcctgaa ggccatcaac ctgatcctgg acgagctgtg gcacaccaac
     2581 gacaaccaga tcgctatctt caaccggctg aagctggtgc ccaagaaggt ggacctgtcc
     2641 cagcagaaag agatccccac caccctggtg gacgacttca tcctgagccc cgtcgtgaag
     2701 agaagcttca tccagagcat caaagtgatc aacgccatca tcaagaagta cggcctgccc
     2761 aacgacatca ttatcgagct ggcccgcgag aagaactcca aggacgccca gaaaatgatc
     2821 aacgagatgc agaagcggaa ccggcagacc aacgagcgga tcgaggaaat catccggacc
     2881 accggcaaag agaacgccaa gtacctgatc gagaagatca agctgcacga catgcaggaa
     2941 ggcaagtgcc tgtacagcct ggaagccatc cctctggaag atctgctgaa caaccccttc
     3001 aactatgagg tggaccacat catccccaga agcgtgtcct tcgacaacag cttcaacaac
     3061 aaggtgctcg tgaagcagga agaagccagc aagaagggca accggacccc attccagtac
     3121 ctgagcagca gcgacagcaa gatcagctac gaaaccttca agaagcacat cctgaatctg
     3181 gccaagggca agggcagaat cagcaagacc aagaaagagt atctgctgga agaacgggac
     3241 atcaacaggt tctccgtgca gaaagacttc atcaaccgga acctggtgga taccagatac
     3301 gccaccagag gcctgatgaa cctgctgcgg agctacttca gagtgaacaa cctggacgtg
     3361 aaagtgaagt ccatcaatgg cggcttcacc agctttctgc ggcggaagtg gaagtttaag
     3421 aaagagcgga acaaggggta caagcaccac gccgaggacg ccctgatcat tgccaacgcc
     3481 gatttcatct tcaaagagtg gaagaaactg gacaaggcca aaaaagtgat ggaaaaccag
     3541 atgttcgagg aaaagcaggc cgagagcatg cccgagatcg aaaccgagca ggagtacaaa
     3601 gagatcttca tcacccccca ccagatcaag cacattaagg acttcaagga ctacaagtac
     3661 agccaccggg tggacaagaa gcctaataga gagctgatta acgacaccct gtactccacc
     3721 cggaaggacg acaagggcaa caccctgatc gtgaacaatc tgaacggcct gtacgacaag
     3781 gacaatgaca agctgaaaaa gctgatcaac aagagccccg aaaagctgct gatgtaccac
     3841 cacgaccccc agacctacca gaaactgaag ctgattatgg aacagtacgg cgacgagaag
     3901 aatcccctgt acaagtacta cgaggaaacc gggaactacc tgaccaagta ctccaaaaag
     3961 gacaacggcc ccgtgatcaa gaagattaag tattacggca acaaactgaa cgcccatctg
     4021 gacatcaccg acgactaccc caacagcaga aacaaggtcg tgaagctgtc cctgaagccc
     4081 tacagattcg acgtgtacct ggacaatggc gtgtacaagt tcgtgaccgt gaagaatctg
     4141 gatgtgatca aaaaagaaaa ctactacgaa gtgaatagca agtgctatga ggaagctaag
     4201 aagctgaaga agatcagcaa ccaggccgag tttatcgcct ccttctacaa caacgatctg
     4261 atcaagatca acggcgagct gtatagagtg atcggcgtga acaacgacct gctgaaccgg
     4321 atcgaagtga acatgatcga catcacctac cgcgagtacc tggaaaacat gaacgacaag
     4381 aggcccccca ggatcattaa gacaatcgcc tccaagaccc agagcattaa gaagtacagc
     4441 acagacattc tgggcaacct gtatgaagtg aaatctaaga agcaccctca gatcatcaaa
     4501 aagggcaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagggatcc
     4561 gaggccagcg gttccggacg ggctgacgca ttggacgatt ttgatctgga tatgctggga
     4621 agtgacgccc tcgatgattt tgaccttgac atgcttggtt cggatgccct tgatgacttt
     4681 gacctcgaca tgctcggcag tgacgccctt gatgatttcg acctggacat gctgattaac
     4741 tctagaagtt ccggatctcc gaaaaagaaa cgcaaagttg gttcgggagg tggttcgggt
     4801 ggctctggat cagtgctgcc tcaggctcct gctcctgcac cagctccagc catggtgtct
     4861 gcactggctc aggcaccagc acccgtgcct gtgctggctc ctggacctcc acaggctgtg
     4921 gctccaccag cccctaaacc tacacaggcc ggcgagggca cactgtctga agctctgctg
     4981 cagctgcagt tcgacgacga ggatctggga gccctgctgg gaaacagcac cgatcctgcc
     5041 gtgttcaccg acctggccag cgtggacaac agcgagttcc agcagctgct gaaccagggc
     5101 atccctgtgg cccctcacac caccgagccc atgctgatgg aataccccga ggccatcacc
     5161 cggctcgtga caggcgctca gaggcctcct gatccagctc ctgcccctct gggagcacca
     5221 ggcctgccta atggactgct gtctggcgac gaggacttca gctctatcgc cgatatggat
     5281 ttctcagcct tgctgtcagg cggtggtagt ggtgggagcg gtagtgacct ttcccatccg
     5341 cccccaaggg gccatctgga tgagctgaca accacacttg agtccatgac cgaggatctg
     5401 aacctggact cacccctgac cccggaattg aacgagattc tggatacctt cctgaacgac
     5461 gagtgcctct tgcatgccat gcatatcagc acaggactgt ccatcttcga cacatctctg
     5521 ttttaggaat tcaaataaaa tacgaaatga aataaaatac gaaatgcaat tgggccgcag
     5581 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     5641 gggcgaccaa aggtcgcccg acgcccgggc tttgcccgag cggcctcagt gagcgagcga
     5701 gcgcgcagct gcctgcagga catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5761 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5821 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5881 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5941 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     6001 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6061 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6121 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6181 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     6241 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6301 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6361 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6421 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6481 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6541 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6601 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     6661 agtgctgcaa tgataccgcg gcttccacgc tcaccggctc cagatttatc agcaataaac
     6721 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6781 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6841 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6901 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6961 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     7021 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7081 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     7141 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7201 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7261 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     7321 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7381 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     7441 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     7501 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     7561 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac
     7621 ggtg
//
