pAAVLINKv2-U6-sgRNA-SCP1-Cre-UDeg3a-3'-SpCas9

LOCUS      pAAVLINKv2-U6-sgRNA-SCP1-Cre-UDeg3a-3'-SpCas9        6712 bp DNA     circular SYN 10-AUG-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6712)
  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..6712
                     /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"
     misc_feature    509..589
                     /label=SCP1
     CDS             601..1629
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     CDS             1645..1734
                     /codon_start=1
                     /label=UDeg3a
                     /translation="LLLKLLLLGGGSGPRPRGTRGKGRRIHYKG"
     misc_feature    1659..1664
                     /label=SDS
     misc_feature    1662..1667
                     /label=SDS
     intron          1738..1778
                     /label=Splicing Donor
     protein_bind    1785..1818
                     /label=lox2272-JTZ17
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     intron          1819..1910
                     /label=Splicing Acceptor
                     /note="Splicing acceptor"
     CDS             1911..3449
                     /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="VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL
                     TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK
                     SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYGV
                     RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG
                     RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDS
                     PTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI
                     IKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ
                     KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLF
                     TLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
     CDS             2052..3449
                     /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="AERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEND
                     KLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLES
                     EFVYGDYKVYGVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIET
                     NGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKK
                     DWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLE
                     AKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY
                     EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP
                     IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI
                     DLSQLGGD"
     CDS             2799..3449
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLII
                     KLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQK
                     QLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT
                     LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
     CDS             3450..3497
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      3501..3932
                     /label=W3SL
                     /note="Short WPRE and SV40 polyA"
     misc_feature    3645..3650
                     /label=SDS
     repeat_region   3975..4115
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     misc_feature    3975..4104
                     /label=AAV2 ITR
     repeat_region   3976..4116
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4190..4645
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        4927..5031
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5032..5892
                     /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      6063..6651
                     /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 gaggtaccgt acttatataa gggggtgggg gcgcgttcgt
      541 cctcagtcgc gatcgaacac tcgagccgag cagacgtgcc tacggaccgc gtacgccacc
      601 atgtccaatt tactgaccgt acaccaaaat ttgcctgcat taccggtcga tgcaacgagt
      661 gatgaggttc gcaagaacct gatggacatg ttcagggatc gccaggcgtt ttctgagcat
      721 acctggaaaa tgcttctgtc cgtttgccgg tcgtgggcgg catggtgcaa gttgaataac
      781 cggaaatggt ttcccgcaga acctgaagat gttcgcgatt atcttctata tcttcaggcg
      841 cgcggtctgg cagtaaaaac catccagcaa catttgggcc agctaaacat gcttcatcgt
      901 cggtccgggc tgccacgacc aagtgacagc aatgctgttt cactggttat gcggcggatc
      961 cgaaaagaaa acgttgatgc cggtgaacgt gcaaaacagg ctctagcgtt cgaacgcact
     1021 gatttcgacc aggttcgttc actcatggaa aatagcgatc gctgccagga tatacgtaat
     1081 ctggcatttc tggggattgc ttataacacc ctgttacgta tagccgaaat tgccaggatc
     1141 agggttaaag atatctcacg tactgacggt gggagaatgt taatccatat tggcagaacg
     1201 aaaacgctgg ttagcaccgc aggtgtagag aaggcactta gcctgggggt aactaaactg
     1261 gtcgagcgat ggatttccgt ctctggtgta gctgatgatc cgaataacta cctgttttgc
     1321 cgggtcagaa aaaatggtgt tgccgcgcca tctgccacca gccagctatc aactcgcgcc
     1381 ctggaaggga tttttgaagc aactcatcga ttgatttacg gcgctaagga tgactctggt
     1441 cagagatacc tggcctggtc tggacacagt gcccgtgtcg gagccgcgcg agatatggcc
     1501 cgcgctggag tttcaatacc ggagatcatg caagctggtg gctggaccaa tgtaaatatt
     1561 gtaatgaact atatccgtaa cctggatagt gaaacagggg caatggtgcg cctgctggaa
     1621 gatggcgatg gaggtggatc tggtctgctg ctgaaactcc tcctcctcgg tggtggttct
     1681 ggtcctcgtc cccgtggtac tcgtggtaaa ggtcgccgta ttcactacaa aggataagta
     1741 agtatcaagg ttacaagaca ggtttaagga gaccaataac tagtataact tcgtatagga
     1801 tactttatag caatttatga aactgggctt gtcgagacag agaagactct tgcgtttctg
     1861 ataggcacct attggtctta ctgacatcca ctttgccttt ctctccacag gtgctgacca
     1921 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
     1981 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
     2041 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
     2101 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
     2161 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
     2221 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
     2281 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
     2341 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacggcgtgc
     2401 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
     2461 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga
     2521 agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
     2581 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
     2641 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
     2701 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
     2761 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
     2821 gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
     2881 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
     2941 ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg
     3001 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
     3061 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
     3121 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
     3181 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
     3241 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
     3301 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
     3361 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
     3421 ggatcgacct gtctcagctg ggaggcgaca aaaggccggc ggccacgaaa aaggccggcc
     3481 aggcaaaaaa gaaaaagtaa ctcgagataa tcaacctctg gattacaaaa tttgtgaaag
     3541 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     3601 gcctttgcat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     3661 ctggttagtt cttgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     3721 ggctcggctg ttgggcactg acaattccgt ggtgtttatt tgtgaaattt gtgatgctat
     3781 tgctttattt gtaaccatct agctttattt gtgaaatttg tgatgctatt gctttatttg
     3841 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     3901 aggttcaggg ggagatgtgg gaggtttttt aaagatctgg agaccggtgt ggcacgtgcg
     3961 gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     4021 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     4081 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     4141 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     4201 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4261 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4321 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4381 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     4441 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     4501 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     4561 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     4621 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     4681 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4741 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4801 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     4861 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     4921 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4981 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     5041 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5101 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5161 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5221 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5281 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5341 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5401 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5461 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5521 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     5581 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5641 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5701 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     5761 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5821 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5881 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     5941 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6001 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6061 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6121 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6181 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6241 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6301 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6361 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6421 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6481 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6541 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6601 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6661 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gt
//