pAAVLINKv1-EF1alpha-Cre-3'-ABE8e

LOCUS       pAAVLINKv1-EF1alpha-Cre-3'-ABE8e               6971 bp DNA     circular SYN 25-NOV-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6971)
  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..6971
                     /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        158..700
                     /label=EF1-alpha
                     /note="Chimeric promoter of ef1a and htlv 5' UTR"
     promoter        187..398
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             411..679
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     misc_feature    505..510
                     /label=SDS
     CDS             714..1745
                     /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"
     intron          1746..1786
                     /label=Splicing Donor
     protein_bind    1791..1824
                     /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          1825..1916
                     /label=Splicing Acceptor
                     /note="Splicing acceptor"
     CDS             1917..3704
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=ABE8e
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="GQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ
                     NGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVV
                     KKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQIL
                     DSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVG
                     TALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLA
                     NGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILP
                     KRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMER
                     SSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPS
                     KYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDK
                     VLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI
                     HQSITGLYETRIDLSQLGGD"
     CDS             2166..3704
                     /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
                     SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV
                     RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG
                     RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDS
                     PTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI
                     IKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ
                     KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLF
                     TLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
     CDS             2307..3704
                     /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
                     EFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIET
                     NGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKK
                     DWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLE
                     AKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY
                     EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP
                     IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI
                     DLSQLGGD"
     CDS             3054..3704
                     /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             3747..3767
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      3771..4202
                     /label=W3SL
                     /note="Short WPRE and SV40 polyA"
     misc_feature    3915..3920
                     /label=SDS
     repeat_region   4245..4374
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #ffe4c4"
     rep_origin      complement(4436..5024)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5195..6055)
                     /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"
     promoter        complement(6056..6160)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6442..6897)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtgtcgac tctagaggat ctgcgatcgc tccggtgccc
      181 gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca
      241 attgaacggg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact
      301 ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga
      361 acgttctttt tcgcaacggg tttgccgcca gaacacagct gaagcttcga ggggctcgca
      421 tctctccttc acgcgcccgc cgccctacct gaggccgcca tccacgccgg ttgagtcgcg
      481 ttctgccgcc tcccgcctgt ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa
      541 agctcaggtc gagaccgggc ctttgtccgg cgctcccttg gagcctacct agactcagcc
      601 ggctctccac gctttgcctg accctgcttg ctcaactcta cgtctttgtt tcgttttctg
      661 ttctgcgccg ttacagatcc aagctgtgac cggcgcctac aacgtacgcc accatgtcca
      721 atttactgac cgtacaccaa aatttgcctg cattaccggt cgatgcaacg agtgatgagg
      781 ttcgcaagaa cctgatggac atgttcaggg atcgccaggc gttttctgag catacctgga
      841 aaatgcttct gtccgtttgc cggtcgtggg cggcatggtg caagttgaat aaccggaaat
      901 ggtttcccgc agaacctgaa gatgttcgcg attatcttct atatcttcag gcgcgcggtc
      961 tggcagtaaa aactatccag caacatttgg gccagctaaa catgcttcat cgtcggtccg
     1021 ggctgccacg accaagtgac agcaatgctg tttcactggt tatgcggcgg atccgaaaag
     1081 aaaacgttga tgccggtgaa cgtgcaaaac aggctctagc gttcgaacgc actgatttcg
     1141 accaggttcg ttcactcatg gaaaatagcg atcgctgcca ggatatacgt aatctggcat
     1201 ttctggggat tgcttataac accctgttac gtatagccga aattgccagg atcagggtta
     1261 aagatatctc acgtactgac ggtgggagaa tgttaatcca tattggcaga acgaaaacgc
     1321 tggttagcac cgcaggtgta gagaaggcac ttagcctggg ggtaactaaa ctggtcgagc
     1381 gatggatttc cgtctctggt gtagctgatg atccgaataa ctacctgttt tgccgggtca
     1441 gaaaaaatgg tgttgccgcg ccatctgcca ccagccagct atcaactcgc gccctggaag
     1501 ggatttttga agcaactcat cgattgattt acggcgctaa ggatgactct ggtcagagat
     1561 acctggcctg gtctggacac agtgcccgtg tcggagccgc gcgagatatg gcccgcgctg
     1621 gagtttcaat accggagatc atgcaagctg gtggctggac caatgtaaat attgtaatga
     1681 actatatccg taacctggat agtgaaacag gggcaatggt gcgcctgctg gaagatggcg
     1741 attaggtaag tatcaaggtt acaagacagg tttaaggaga ccaatagctt ataacttcgt
     1801 ataggatact ttatagcaat ttatgaaact gggcttgtcg agacagagaa gactcttgcg
     1861 tttctgatag gcacctattg gtcttactga catccacttt gcctttctct ccacagggac
     1921 agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc
     1981 agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt
     2041 actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt
     2101 ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca
     2161 acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag
     2221 aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc
     2281 agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg
     2341 ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga
     2401 tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga
     2461 aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca
     2521 aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg
     2581 gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca
     2641 aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg
     2701 ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca
     2761 acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt
     2821 gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata
     2881 tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga
     2941 ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct
     3001 tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt
     3061 ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct
     3121 tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc
     3181 tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc
     3241 tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga
     3301 acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc
     3361 agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca
     3421 gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct
     3481 acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta
     3541 ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga
     3601 agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg
     3661 gcctgtacga gacacggatc gacctgtctc agctgggagg tgactctggc ggctcaaaaa
     3721 gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtctaa ctcgagataa
     3781 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     3841 ttttacgcta tgtggatacg ctgctttaat gcctttgcat catgctattg cttcccgtat
     3901 ggctttcatt ttctcctcct tgtataaatc ctggttagtt cttgccacgg cggaactcat
     3961 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     4021 ggtgtttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatct agctttattt
     4081 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     4141 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt
     4201 aaagatctgg agaccggtgt ggcacgtgcg gaccgagcgg ccgcaggaac ccctagtgat
     4261 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     4321 cgcccgacgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggacatgt
     4381 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4441 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4501 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4561 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4621 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4681 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4741 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4801 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4861 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4921 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4981 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5041 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5101 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5161 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5221 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5281 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5341 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5401 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5461 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5521 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5581 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5641 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     5701 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     5761 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     5821 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     5881 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     5941 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6001 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6061 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6121 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6181 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     6241 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     6301 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     6361 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     6421 ttgtactgag agtgcaccat aaaattgtaa acgttaatat tttgttaaaa ttcgcgttaa
     6481 atttttgtta aatcagctca ttttttaacc aatagaccga aatcggcaaa atcccttata
     6541 aatcaaaaga atagcccgag atagagttga gtgttgttcc agtttggaac aagagtccac
     6601 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     6661 cactacgtga accatcaccc aaatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     6721 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgagcgtgg
     6781 cgagaaagga agggaagaaa gcgaaaggag cgggcgctaa ggcgctggca agtgtagcgg
     6841 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtact
     6901 atggttgctt tgacgtatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     6961 catcaggcgc c
//