pAAVLINK-U6-sgRNA-EF1alpha-5'-dSpCas9-VPH

LOCUS       pAAVLINK-U6-sgRNA-EF1alpha-5'-dSpCas9-VPH               7353 bp DNA     circular SYN 22-AUG-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7353)
  AUTHORS   Stavrini Papadaki
  TITLE     Direct Submission
  JOURNAL   Exported Jun 15, 2026 from SnapGene 6.0.2
            https://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..7353
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        61..309
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        61..301
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        331..406
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        420..962
                     /label=EF1-alpha
                     /note="Chimeric promoter of ef1a and htlv 5' UTR"
     promoter        449..660
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             673..941
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     misc_feature    767..772
                     /label=SDS
     CDS             978..1004
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1008..1028
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1038..3740
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTK"
     CDS             1038..2753
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     intron          4488..4528
                     /label=Splicing Donor
     misc_feature    4538..4571
                     /label=lox2272-JT15
     repeat_region   4616..4756
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     misc_feature    4616..4745
                     /label=AAV2 ITR
     repeat_region   4617..4757
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4831..5286
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        5568..5672
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5673..6533
                     /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"
     rep_origin      6704..7292
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctgcaggca gagagggagt ggccaactcc atcactaggg gttcctgcgg ccgcacgcgt
       61 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
      121 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      181 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      241 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      301 cgaaacaccg taagtcaata gttccatgat gttttagagc tagaaatagc aagttaaaat
      361 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttgtacagg
      421 atctgcgatc gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
      481 aagttggggg gaggggtcgg caattgaacg ggtgcctaga gaaggtggcg cggggtaaac
      541 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
      601 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
      661 ctgaagcttc gaggggctcg catctctcct tcacgcgccc gccgccctac ctgaggccgc
      721 catccacgcc ggttgagtcg cgttctgccg cctcccgcct gtggtgcctc ctgaactgcg
      781 tccgccgtct aggtaagttt aaagctcagg tcgagaccgg gcctttgtcc ggcgctccct
      841 tggagcctac ctagactcag ccggctctcc acgctttgcc tgaccctgct tgctcaactc
      901 tacgtctttg tttcgttttc tgttctgcgc cgttacagat ccaagctgtg accggcgcct
      961 accgtacggc caccatgtac ccatacgatg ttccagatta cgcttcgccg aagaaaaagc
     1021 gcaaggtcga agcgtccgac aagaagtaca gcatcggcct ggccatcggc accaactctg
     1081 tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg
     1141 gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg
     1201 gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga
     1261 agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca
     1321 gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc
     1381 accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct
     1441 accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc
     1501 tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc
     1561 ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt
     1621 tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac
     1681 tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg
     1741 gcctgttcgg caacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact
     1801 tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg
     1861 acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc
     1921 tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc
     1981 ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga
     2041 aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca
     2101 agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca
     2161 tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag
     2221 aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc
     2281 tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca
     2341 accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg
     2401 ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct
     2461 ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga
     2521 ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg
     2581 agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa
     2641 agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca
     2701 accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg
     2761 actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg
     2821 atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc
     2881 tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc
     2941 tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat
     3001 acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg
     3061 gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc
     3121 tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc
     3181 agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg
     3241 gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc
     3301 ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga
     3361 acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc
     3421 tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc
     3481 tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact
     3541 acgatgtgga ccacatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg
     3601 tgctgaccag aagcgacaag gcccggggca agagcgacaa cgtgccctcc gaagaggtcg
     3661 tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa
     3721 agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct
     3781 tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg
     3841 actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga
     3901 tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc
     3961 gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg
     4021 ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt
     4081 acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt
     4141 acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg
     4201 agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata
     4261 agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga
     4321 aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca
     4381 gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca
     4441 gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaaggta agtatcaagg
     4501 ttacaagaca ggtttaagga gaccaataag cgctagcaat tattcgtata ggatacttta
     4561 tacgaagtta ttaagttatg gagaccggtg tggcacgtgc ggaccgagcg gccgcaggaa
     4621 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     4681 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     4741 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4801 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
     4861 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc
     4921 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     4981 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     5041 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     5101 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     5161 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt
     5221 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     5281 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     5341 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     5401 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     5461 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     5521 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5581 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5641 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5701 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5761 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5821 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5881 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     5941 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     6001 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     6061 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     6121 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     6181 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     6241 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     6301 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     6361 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     6421 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     6481 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     6541 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6601 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6661 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6721 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6781 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6841 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6901 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6961 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     7021 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     7081 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     7141 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     7201 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     7261 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     7321 tcctggcctt ttgctggcct tttgctcaca tgt
//