pAAVLINK-U6-sgRNA-EF1alpha-5'-SpCas9

LOCUS       pAAVLINK-U6-sgRNA-EF1alpha-5'-SpCas9          6602 bp DNA     circular SYN 07-APR-2026
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6602)
  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..6602
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #ffe4c4"
     promoter        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        145..385
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        415..490
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        539..750
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             763..1031
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      1045..1058
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1058..1123
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1130..1150
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1175..3736
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK"
     CDS             1175..2890
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     intron          3737..3777
                     /label=Splicing Donor
     misc_feature    3787..3820
                     /label=lox2272-JT15
     repeat_region   3865..4005
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     misc_feature    3865..3994
                     /label=AAV2 ITR
     repeat_region   3866..4006
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4080..4535
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        4817..4921
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4922..5782
                     /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      5953..6541
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accgactcca gtctttctag aagagtttta
      421 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
      481 gagtcggtgc tttttttcta gaggtaccgg atctgcgatc gctccggtgc ccgtcagtgg
      541 gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacg
      601 ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
      661 ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt
      721 tttcgcaacg ggtttgccgc cagaacacag ctgaagcttc gaggggctcg catctctcct
      781 tcacgcgccc gccgccctac ctgaggccgc catccacgcc ggttgagtcg cgttctgccg
      841 cctcccgcct gtggtgcctc ctgaactgcg tccgccgtct aggtaagttt aaagctcagg
      901 tcgagaccgg gcctttgtcc ggcgctccct tggagcctac ctagactcag ccggctctcc
      961 acgctttgcc tgaccctgct tgctcaactc tacgtctttg tttcgttttc tgttctgcgc
     1021 cgttacagat ccaagctgtg accggcgcct acccatggac tataaggacc acgacggaga
     1081 ctacaaggat catgatattg attacaaaga cgatgacgat aagatggccc caaagaagaa
     1141 gcggaaggtc ggtatccacg gagtcccagc agccgacaag aagtacagca tcggcctgga
     1201 catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa
     1261 gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc
     1321 cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag
     1381 aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat
     1441 ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga
     1501 taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga
     1561 gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga
     1621 cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat
     1681 cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca
     1741 gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc
     1801 catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc
     1861 cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc
     1921 caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac
     1981 ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt
     2041 tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac
     2101 cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca
     2161 ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat
     2221 tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga
     2281 agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct
     2341 cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat
     2401 cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta
     2461 cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta
     2521 ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga
     2581 ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag
     2641 cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa
     2701 gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt
     2761 gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga
     2821 cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa
     2881 gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc
     2941 cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga
     3001 ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga
     3061 gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca
     3121 gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat
     3181 ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa
     3241 cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa
     3301 agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag
     3361 ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt
     3421 gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac
     3481 ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga
     3541 gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa
     3601 gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat
     3661 caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga
     3721 ctccatcgac aacaaggtaa gtatcaaggt tacaagacag gtttaaggag accaataagc
     3781 gctagcaatt attcgtatag gatactttat acgaagttat taagttatgg agaccggtgt
     3841 ggcacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc
     3901 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     3961 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     4021 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     4081 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4141 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4201 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     4261 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     4321 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4381 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
     4441 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4501 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     4561 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     4621 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     4681 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     4741 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     4801 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4861 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4921 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     4981 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5041 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5101 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5161 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5221 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5281 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5341 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5401 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     5461 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     5521 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     5581 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     5641 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     5701 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5761 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     5821 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     5881 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     5941 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6001 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6061 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6121 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6181 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6241 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6301 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6361 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6421 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     6481 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     6541 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     6601 gt
//