LOCUS       sequence-317304-        7729 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutagenesis of Th.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7729)
  TITLE     Zweifel CRISPR AAV Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7729)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-CMV-FLEX-SaCas9-U6-sgTh
FEATURES             Location/Qualifiers
     source          1..7729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    complement(621..654)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(705..738)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(750..830)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(837..884)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(885..4040)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             complement(4065..4085)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    4117..4150
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    4201..4234
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4259..4276)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4265..4472
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4479..4727
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4479..4499
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4650..4669
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4750..4825
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4839..4979
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4839..4968
                     /label=AAV2 ITR
     rep_origin      5054..5509
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5141..5160)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5351..5372
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5526..5545)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5645..5667
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5705..5723)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5791..5895
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5896..5964,5965..6756)
                     /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     complement(6114..6133)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6927..7515
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7416..7435
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7577..7706
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactaccg
      601 gtgctagcgt cgactccgga ataacttcgt ataggatact ttatacgaag ttatgcagaa
      661 tggtagctgg attgtagctg ctattagcaa tatgaaacct cttaataact tcgtatagca
      721 tacattatac gaagttatgg cgcgccttaa gcgtaatctg gaacatcgta tgggtaagcg
      781 taatctggaa catcgtatgg gtaagcgtaa tctggaacat cgtatgggta ggatcccttt
      841 ttcttttttg cctggccggc ctttttcgtg gccgccggcc ttttgccctt tttgatgatc
      901 tgagggtgct tcttagattt cacttcatac aggttgccca gaatgtctgt gctgtacttc
      961 ttaatgctct gggtcttgga ggcgattgtc ttaatgatcc tggggggcct cttgtcgttc
     1021 atgttttcca ggtactcgcg gtaggtgatg tcgatcatgt tcacttcgat ccggttcagc
     1081 aggtcgttgt tcacgccgat cactctatac agctcgccgt tgatcttgat cagatcgttg
     1141 ttgtagaagg aggcgataaa ctcggcctgg ttgctgatct tcttcagctt cttagcttcc
     1201 tcatagcact tgctattcac ttcgtagtag ttttcttttt tgatcacatc cagattcttc
     1261 acggtcacga acttgtacac gccattgtcc aggtacacgt cgaatctgta gggcttcagg
     1321 gacagcttca cgaccttgtt tctgctgttg gggtagtcgt cggtgatgtc cagatgggcg
     1381 ttcagtttgt tgccgtaata cttaatcttc ttgatcacgg ggccgttgtc ctttttggag
     1441 tacttggtca ggtagttccc ggtttcctcg tagtacttgt acaggggatt cttctcgtcg
     1501 ccgtactgtt ccataatcag cttcagtttc tggtaggtct gggggtcgtg gtggtacatc
     1561 agcagctttt cggggctctt gttgatcagc tttttcagct tgtcattgtc cttgtcgtac
     1621 aggccgttca gattgttcac gatcagggtg ttgcccttgt cgtccttccg ggtggagtac
     1681 agggtgtcgt taatcagctc tctattaggc ttcttgtcca cccggtggct gtacttgtag
     1741 tccttgaagt ccttaatgtg cttgatctgg tggggggtga tgaagatctc tttgtactcc
     1801 tgctcggttt cgatctcggg catgctctcg gcctgctttt cctcgaacat ctggttttcc
     1861 atcacttttt tggccttgtc cagtttcttc cactctttga agatgaaatc ggcgttggca
     1921 atgatcaggg cgtcctcggc gtggtgcttg taccccttgt tccgctcttt cttaaacttc
     1981 cacttccgcc gcagaaagct ggtgaagccg ccattgatgg acttcacttt cacgtccagg
     2041 ttgttcactc tgaagtagct ccgcagcagg ttcatcaggc ctctggtggc gtatctggta
     2101 tccaccaggt tccggttgat gaagtctttc tgcacggaga acctgttgat gtcccgttct
     2161 tccagcagat actctttctt ggtcttgctg attctgccct tgcccttggc cagattcagg
     2221 atgtgcttct tgaaggtttc gtagctgatc ttgctgtcgc tgctgctcag gtactggaat
     2281 ggggtccggt tgcccttctt gctgttttct tcctgcttca cgagcacctt gttgttgaag
     2341 ctgttgtcga aggacacgct tctggggatg atgtggtcca cctcatagtt gaaggggttg
     2401 ttcagcagat cttccagagg gatggcttcc aggctgtaca ggcacttgcc ttcctgcatg
     2461 tcgtgcagct tgatcttctc gatcaggtac ttggcgttct ctttgccggt ggtccggatg
     2521 atttcctcga tccgctcgtt ggtctgccgg ttccgcttct gcatctcgtt gatcattttc
     2581 tgggcgtcct tggagttctt ctcgcgggcc agctcgataa tgatgtcgtt gggcaggccg
     2641 tacttcttga tgatggcgtt gatcactttg atgctctgga tgaagcttct cttcacgacg
     2701 gggctcagga tgaagtcgtc caccagggtg gtggggatct ctttctgctg ggacaggtcc
     2761 accttcttgg gcaccagctt cagccggttg aagatagcga tctggttgtc gttggtgtgc
     2821 cacagctcgt ccaggatcag gttgatggcc ttcaggctca ggttgtgggt gccggtatag
     2881 cccttcagat tagagatctg ctcgatctct tcctgggtca gctcggagtt cagattggtc
     2941 agttcttcct ggatgtcctc gctgctctgg tagatggtca ggatcttggc aatctgatcc
     3001 agcagctcgg cgttctcaat aatctctttc cgggcggtaa tgtccttgat gtcgtggtac
     3061 accttcaggt tggtgaactc gggcttgccg gtgctggtca ctctgtagcc cttaatatcc
     3121 tcttcgttca cgaggatttc tttggcgatc tgcttcaggg tgggcttctt cttctgcttg
     3181 aacacgttct cgatgatctg gaacttctcg taatattcca gcttctcgtt ctcgtccctg
     3241 gtgatcacga gattgttcag gtcgttcagg gcgttgtaca ggtcggcgtt gtaggcgtac
     3301 ttcacgctcc gcagttcctc ggggaagtag gtgcagtggc ccatcagcat ctcgtaccat
     3361 tctttgatgt ccttccagcc gaaggggctg ccctcgccag gtccctcata gtaggtccgc
     3421 cgggtttcca gcaggtcgat gtaggtgtcg atgaagctct ggtccagctg gtggtaggcc
     3481 ttctgcacct tcagcagctg tttggcttct ttcacgtagt cgctggtctt gaatctgttg
     3541 atgctgcccc gcacttcgcc gtctttcttc agccgttcca gctgcagttc ggccacgtat
     3601 ttctcttcca gggccttgct gttccggctg atctgctctt tggtggacag ctcgttgccg
     3661 gtgtcctctt ccacctcgtt cacgttgtgc acgcctcttc tcttggccag gtgcagcagg
     3721 gcggcagaga actcttcctc gctcagcttc tggctcaggc ccttcactct ggcctcgtag
     3781 gggttgatgc cgctcagctc gctgtggtcg gtcagcaggt tgtagtcgaa cagcagcttc
     3841 ttcactctct ggattctatg ccgcctccgc cgcttcagcc ttctggcgcc tctcttgctc
     3901 cgcctgccct cgttgttttc cacgttggcc tctttgaaca gccgcacgcc ggcatcgatc
     3961 acgtcccgtg tctcgtagtc gatgatgccg tagcccacgc tggtgatgcc gatgtccagg
     4021 cccaggatgt agttccgctt ggctgctggg actccgtgga taccgacctt ccgcttcttc
     4081 tttggggcca tgcggccgct gcagaccggt gctagcataa cttcgtataa agtatcctat
     4141 acgaagttat ttgccttaac ccagaaatta tcactgttat tctttagaat ggtgcaaaga
     4201 ataacttcgt ataatgtatg ctatacgaag ttatgaattc ctagagctcg ctgatcagcc
     4261 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     4321 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     4381 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     4441 gattgggaag agaatagcag gcatgctggg gaggtaccga gggcctattt cccatgattc
     4501 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
     4561 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     4621 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     4681 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgcc aaggttcatt
     4741 ggacggcggg ttttagtact ctggaaacag aatctactaa aacaaggcaa aatgccgtgt
     4801 ttatctcgtc aacttgttgg cgagattttt gcggccgcag gaacccctag tgatggagtt
     4861 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     4921 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg
     4981 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     5041 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     5101 cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct
     5161 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     5221 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     5281 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     5341 ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat
     5401 tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt
     5461 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     5521 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     5581 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     5641 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     5701 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     5761 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5821 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     5881 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5941 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     6001 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     6061 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     6121 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     6181 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     6241 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     6301 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     6361 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     6421 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     6481 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     6541 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     6601 ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     6661 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     6721 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6781 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6841 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6901 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6961 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     7021 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     7081 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     7141 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     7201 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     7261 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     7321 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     7381 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     7441 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     7501 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     7561 ccttttgctc acatgtcctg caggcagctg cgcgctcgct cgctcactga ggccgcccgg
     7621 gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt
     7681 ggccaactcc atcactaggg gttcctgcgg cctctagact cgaggcgtt
//
