LOCUS       sequence-481798-        7448 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Cre-KO AAV vector#1 containing SaCas9 and its sgRNA targeting Cre.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7448)
  TITLE     Ultrabright Chemical Labeling Enables Rapid Neural Connectivity 
            Profiling in Large Tissue Samples
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7448)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 7, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pX601-AAV-CMV-NLS-SaCas9-NLS-3xHA-bGHpA;U6-Cre-1
FEATURES             Location/Qualifiers
     source          1..7448
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2313..2360
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             2367..2447
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(2475..2492)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2481..2688
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2695..2943
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2695..2715
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2866..2885
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2966..3041
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   3055..3195
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3055..3184
                     /label=AAV2 ITR
     rep_origin      3270..3725
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3357..3376)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3567..3588
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3742..3761)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3861..3883
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3921..3939)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4007..4111
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4112..4180,4181..4972)
                     /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(4330..4349)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5143..5731
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5632..5651
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5793..5922
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        5946..6325
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6326..6529
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6480..6500
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      6548..6557
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6560..6580
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(6605..7448,1..2312)
                     /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"
ORIGIN
        1 tcatcgagaa cgtgttcaag cagaagaaga agcccaccct gaagcagatc gccaaagaaa
       61 tcctcgtgaa cgaagaggat attaagggct acagagtgac cagcaccggc aagcccgagt
      121 tcaccaacct gaaggtgtac cacgacatca aggacattac cgcccggaaa gagattattg
      181 agaacgccga gctgctggat cagattgcca agatcctgac catctaccag agcagcgagg
      241 acatccagga agaactgacc aatctgaact ccgagctgac ccaggaagag atcgagcaga
      301 tctctaatct gaagggctat accggcaccc acaacctgag cctgaaggcc atcaacctga
      361 tcctggacga gctgtggcac accaacgaca accagatcgc tatcttcaac cggctgaagc
      421 tggtgcccaa gaaggtggac ctgtcccagc agaaagagat ccccaccacc ctggtggacg
      481 acttcatcct gagccccgtc gtgaagagaa gcttcatcca gagcatcaaa gtgatcaacg
      541 ccatcatcaa gaagtacggc ctgcccaacg acatcattat cgagctggcc cgcgagaaga
      601 actccaagga cgcccagaaa atgatcaacg agatgcagaa gcggaaccgg cagaccaacg
      661 agcggatcga ggaaatcatc cggaccaccg gcaaagagaa cgccaagtac ctgatcgaga
      721 agatcaagct gcacgacatg caggaaggca agtgcctgta cagcctggaa gccatccctc
      781 tggaagatct gctgaacaac cccttcaact atgaggtgga ccacatcatc cccagaagcg
      841 tgtccttcga caacagcttc aacaacaagg tgctcgtgaa gcaggaagaa aacagcaaga
      901 agggcaaccg gaccccattc cagtacctga gcagcagcga cagcaagatc agctacgaaa
      961 ccttcaagaa gcacatcctg aatctggcca agggcaaggg cagaatcagc aagaccaaga
     1021 aagagtatct gctggaagaa cgggacatca acaggttctc cgtgcagaaa gacttcatca
     1081 accggaacct ggtggatacc agatacgcca ccagaggcct gatgaacctg ctgcggagct
     1141 acttcagagt gaacaacctg gacgtgaaag tgaagtccat caatggcggc ttcaccagct
     1201 ttctgcggcg gaagtggaag tttaagaaag agcggaacaa ggggtacaag caccacgccg
     1261 aggacgccct gatcattgcc aacgccgatt tcatcttcaa agagtggaag aaactggaca
     1321 aggccaaaaa agtgatggaa aaccagatgt tcgaggaaaa gcaggccgag agcatgcccg
     1381 agatcgaaac cgagcaggag tacaaagaga tcttcatcac cccccaccag atcaagcaca
     1441 ttaaggactt caaggactac aagtacagcc accgggtgga caagaagcct aatagagagc
     1501 tgattaacga caccctgtac tccacccgga aggacgacaa gggcaacacc ctgatcgtga
     1561 acaatctgaa cggcctgtac gacaaggaca atgacaagct gaaaaagctg atcaacaaga
     1621 gccccgaaaa gctgctgatg taccaccacg acccccagac ctaccagaaa ctgaagctga
     1681 ttatggaaca gtacggcgac gagaagaatc ccctgtacaa gtactacgag gaaaccggga
     1741 actacctgac caagtactcc aaaaaggaca acggccccgt gatcaagaag attaagtatt
     1801 acggcaacaa actgaacgcc catctggaca tcaccgacga ctaccccaac agcagaaaca
     1861 aggtcgtgaa gctgtccctg aagccctaca gattcgacgt gtacctggac aatggcgtgt
     1921 acaagttcgt gaccgtgaag aatctggatg tgatcaaaaa agaaaactac tacgaagtga
     1981 atagcaagtg ctatgaggaa gctaagaagc tgaagaagat cagcaaccag gccgagttta
     2041 tcgcctcctt ctacaacaac gatctgatca agatcaacgg cgagctgtat agagtgatcg
     2101 gcgtgaacaa cgacctgctg aaccggatcg aagtgaacat gatcgacatc acctaccgcg
     2161 agtacctgga aaacatgaac gacaagaggc cccccaggat cattaagaca atcgcctcca
     2221 agacccagag cattaagaag tacagcacag acattctggg caacctgtat gaagtgaaat
     2281 ctaagaagca ccctcagatc atcaaaaagg gcaaaaggcc ggcggccacg aaaaaggccg
     2341 gccaggcaaa aaagaaaaag ggatcctacc catacgatgt tccagattac gcttacccat
     2401 acgatgttcc agattacgct tacccatacg atgttccaga ttacgcttaa gaattcctag
     2461 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     2521 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     2581 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     2641 ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagg taccgagggc
     2701 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
     2761 ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
     2821 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
     2881 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac
     2941 accgcctgca ttaccggtcg atgcagtttt agtactctgg aaacagaatc tactaaaaca
     3001 aggcaaaatg ccgtgtttat ctcgtcaact tgttggcgag atttttgcgg ccgcaggaac
     3061 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     3121 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     3181 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3241 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     3301 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc
     3361 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3421 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3481 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3541 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3601 ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt
     3661 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     3721 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     3781 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     3841 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     3901 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     3961 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4021 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4081 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     4141 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     4201 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     4261 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     4321 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     4381 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     4441 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     4501 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     4561 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     4621 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     4681 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     4741 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     4801 tgataaatct ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga
     4861 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     4921 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     4981 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5041 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     5101 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5161 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5221 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5281 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5341 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5401 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5461 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5521 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5581 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5641 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5701 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5761 cctggccttt tgctggcctt ttgctcacat gtcctgcagg cagctgcgcg ctcgctcgct
     5821 cactgaggcc gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag
     5881 cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggcctc tagactcgag
     5941 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
     6001 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
     6061 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     6121 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     6181 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     6241 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     6301 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
     6361 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
     6421 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
     6481 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac
     6541 taccggtgcc accatggccc caaagaagaa gcggaaggtc ggtatccacg gagtcccagc
     6601 agccaagcgg aactacatcc tgggcctgga catcggcatc accagcgtgg gctacggcat
     6661 catcgactac gagacacggg acgtgatcga tgccggcgtg cggctgttca aagaggccaa
     6721 cgtggaaaac aacgagggca ggcggagcaa gagaggcgcc agaaggctga agcggcggag
     6781 gcggcataga atccagagag tgaagaagct gctgttcgac tacaacctgc tgaccgacca
     6841 cagcgagctg agcggcatca acccctacga ggccagagtg aagggcctga gccagaagct
     6901 gagcgaggaa gagttctctg ccgccctgct gcacctggcc aagagaagag gcgtgcacaa
     6961 cgtgaacgag gtggaagagg acaccggcaa cgagctgtcc accaaagagc agatcagccg
     7021 gaacagcaag gccctggaag agaaatacgt ggccgaactg cagctggaac ggctgaagaa
     7081 agacggcgaa gtgcggggca gcatcaacag attcaagacc agcgactacg tgaaagaagc
     7141 caaacagctg ctgaaggtgc agaaggccta ccaccagctg gaccagagct tcatcgacac
     7201 ctacatcgac ctgctggaaa cccggcggac ctactatgag ggacctggcg agggcagccc
     7261 cttcggctgg aaggacatca aagaatggta cgagatgctg atgggccact gcacctactt
     7321 ccccgaggaa ctgcggagcg tgaagtacgc ctacaacgcc gacctgtaca acgccctgaa
     7381 cgacctgaac aatctcgtga tcaccaggga cgagaacgag aagctggaat attacgagaa
     7441 gttccaga
//
