LOCUS       sequence-481799-        7449 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Cre-KO AAV vector#2 containing SaCas9 and its sgRNA targeting Cre.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7449)
  TITLE     Ultrabright Chemical Labeling Enables Rapid Neural Connectivity 
            Profiling in Large Tissue Samples
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7449)
  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            "Unpublished"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-2
FEATURES             Location/Qualifiers
     source          1..7449
                     /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        2967..3042
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   3056..3196
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3056..3185
                     /label=AAV2 ITR
     rep_origin      3271..3726
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3358..3377)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3568..3589
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3743..3762)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3862..3884
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3922..3940)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4008..4112
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4113..4181,4182..4973)
                     /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(4331..4350)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5144..5732
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5633..5652
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5794..5923
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        5947..6326
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6327..6530
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6481..6501
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      6549..6558
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6561..6581
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(6606..7449,1..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 accgatgctg tttcactggt tatgcggttt tagtactctg gaaacagaat ctactaaaac
     3001 aaggcaaaat gccgtgttta tctcgtcaac ttgttggcga gatttttgcg gccgcaggaa
     3061 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     3121 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     3181 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3241 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
     3301 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc
     3361 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3421 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3481 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     3541 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     3601 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt
     3661 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     3721 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     3781 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3841 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3901 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     3961 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     4021 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     4081 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     4141 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     4201 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     4261 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     4321 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     4381 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     4441 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     4501 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     4561 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     4621 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     4681 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     4741 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     4801 ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca ctggggccag
     4861 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     4921 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     4981 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     5041 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     5101 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     5161 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     5221 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     5281 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     5341 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     5401 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     5461 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     5521 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     5581 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     5641 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     5701 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     5761 tcctggcctt ttgctggcct tttgctcaca tgtcctgcag gcagctgcgc gctcgctcgc
     5821 tcactgaggc cgcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga
     5881 gcgcgcagag agggagtggc caactccatc actaggggtt cctgcggcct ctagactcga
     5941 ggcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
     6001 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     6061 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     6121 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     6181 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
     6241 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
     6301 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
     6361 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
     6421 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
     6481 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa
     6541 ctaccggtgc caccatggcc ccaaagaaga agcggaaggt cggtatccac ggagtcccag
     6601 cagccaagcg gaactacatc ctgggcctgg acatcggcat caccagcgtg ggctacggca
     6661 tcatcgacta cgagacacgg gacgtgatcg atgccggcgt gcggctgttc aaagaggcca
     6721 acgtggaaaa caacgagggc aggcggagca agagaggcgc cagaaggctg aagcggcgga
     6781 ggcggcatag aatccagaga gtgaagaagc tgctgttcga ctacaacctg ctgaccgacc
     6841 acagcgagct gagcggcatc aacccctacg aggccagagt gaagggcctg agccagaagc
     6901 tgagcgagga agagttctct gccgccctgc tgcacctggc caagagaaga ggcgtgcaca
     6961 acgtgaacga ggtggaagag gacaccggca acgagctgtc caccaaagag cagatcagcc
     7021 ggaacagcaa ggccctggaa gagaaatacg tggccgaact gcagctggaa cggctgaaga
     7081 aagacggcga agtgcggggc agcatcaaca gattcaagac cagcgactac gtgaaagaag
     7141 ccaaacagct gctgaaggtg cagaaggcct accaccagct ggaccagagc ttcatcgaca
     7201 cctacatcga cctgctggaa acccggcgga cctactatga gggacctggc gagggcagcc
     7261 ccttcggctg gaaggacatc aaagaatggt acgagatgct gatgggccac tgcacctact
     7321 tccccgagga actgcggagc gtgaagtacg cctacaacgc cgacctgtac aacgccctga
     7381 acgacctgaa caatctcgtg atcaccaggg acgagaacga gaagctggaa tattacgaga
     7441 agttccaga
//
