LOCUS       sequence-418181-        7728 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 7728)
  AUTHORS   Yu W, Pati D, Pina MM, Schmidt KT, Boyt KM, Hunker AC, Zweifel LS, 
            McElligott ZA, Kash TL
  TITLE     Periaqueductal gray/dorsal raphe dopamine neurons contribute to sex 
            differences in pain-related behaviors.
  JOURNAL   Neuron. 2021 Apr 21;109(8):1365-1380.e5. doi: 
            10.1016/j.neuron.2021.03.001. Epub 2021 Mar 18.
  PUBMED    33740416
REFERENCE   2  (bases 1 to 7728)
  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"; doi: 
            "10.1016/j.neuron.2021.03.001"; journalName: "Neuron"; date: 
            "2021-04-21- 21"; volume: "109"; issue: "8"; pages: "1365-1380.e5"
COMMENT     Sequence Label: pAAV-CMV-FLEX-SaCas9-U6-sgTh(2)
FEATURES             Location/Qualifiers
     source          1..7728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        24..403
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        404..607
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     558..578
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    complement(644..677)
                     /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(728..761)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(773..853)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(860..907)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(908..4063)
                     /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(4088..4108)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    4140..4173
                     /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    4224..4257
                     /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(4282..4299)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4288..4495
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4502..4750
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4502..4522
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4673..4692
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4772..4847
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4861..5001
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4861..4990
                     /label=AAV2 ITR
     rep_origin      5076..5531
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5163..5182)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5373..5394
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5548..5567)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5667..5689
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5727..5745)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5813..5917
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5918..5986,5987..6778)
                     /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(6136..6155)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6949..7537
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7438..7457
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7599..7728
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gcggcctcta gactcgaggc gttgacattg attattgact agttattaat agtaatcaat
       61 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
      121 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
      181 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
      241 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
      301 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
      361 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
      421 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
      481 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
      541 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
      601 cagagctctc tggctaacta ccggtgctag cgtcgactcc ggaataactt cgtataggat
      661 actttatacg aagttatgca gaatggtagc tggattgtag ctgctattag caatatgaaa
      721 cctcttaata acttcgtata gcatacatta tacgaagtta tggcgcgcct taagcgtaat
      781 ctggaacatc gtatgggtaa gcgtaatctg gaacatcgta tgggtaagcg taatctggaa
      841 catcgtatgg gtaggatccc tttttctttt ttgcctggcc ggcctttttc gtggccgccg
      901 gccttttgcc ctttttgatg atctgagggt gcttcttaga tttcacttca tacaggttgc
      961 ccagaatgtc tgtgctgtac ttcttaatgc tctgggtctt ggaggcgatt gtcttaatga
     1021 tcctgggggg cctcttgtcg ttcatgtttt ccaggtactc gcggtaggtg atgtcgatca
     1081 tgttcacttc gatccggttc agcaggtcgt tgttcacgcc gatcactcta tacagctcgc
     1141 cgttgatctt gatcagatcg ttgttgtaga aggaggcgat aaactcggcc tggttgctga
     1201 tcttcttcag cttcttagct tcctcatagc acttgctatt cacttcgtag tagttttctt
     1261 ttttgatcac atccagattc ttcacggtca cgaacttgta cacgccattg tccaggtaca
     1321 cgtcgaatct gtagggcttc agggacagct tcacgacctt gtttctgctg ttggggtagt
     1381 cgtcggtgat gtccagatgg gcgttcagtt tgttgccgta atacttaatc ttcttgatca
     1441 cggggccgtt gtcctttttg gagtacttgg tcaggtagtt cccggtttcc tcgtagtact
     1501 tgtacagggg attcttctcg tcgccgtact gttccataat cagcttcagt ttctggtagg
     1561 tctgggggtc gtggtggtac atcagcagct tttcggggct cttgttgatc agctttttca
     1621 gcttgtcatt gtccttgtcg tacaggccgt tcagattgtt cacgatcagg gtgttgccct
     1681 tgtcgtcctt ccgggtggag tacagggtgt cgttaatcag ctctctatta ggcttcttgt
     1741 ccacccggtg gctgtacttg tagtccttga agtccttaat gtgcttgatc tggtgggggg
     1801 tgatgaagat ctctttgtac tcctgctcgg tttcgatctc gggcatgctc tcggcctgct
     1861 tttcctcgaa catctggttt tccatcactt ttttggcctt gtccagtttc ttccactctt
     1921 tgaagatgaa atcggcgttg gcaatgatca gggcgtcctc ggcgtggtgc ttgtacccct
     1981 tgttccgctc tttcttaaac ttccacttcc gccgcagaaa gctggtgaag ccgccattga
     2041 tggacttcac tttcacgtcc aggttgttca ctctgaagta gctccgcagc aggttcatca
     2101 ggcctctggt ggcgtatctg gtatccacca ggttccggtt gatgaagtct ttctgcacgg
     2161 agaacctgtt gatgtcccgt tcttccagca gatactcttt cttggtcttg ctgattctgc
     2221 ccttgccctt ggccagattc aggatgtgct tcttgaaggt ttcgtagctg atcttgctgt
     2281 cgctgctgct caggtactgg aatggggtcc ggttgccctt cttgctgttt tcttcctgct
     2341 tcacgagcac cttgttgttg aagctgttgt cgaaggacac gcttctgggg atgatgtggt
     2401 ccacctcata gttgaagggg ttgttcagca gatcttccag agggatggct tccaggctgt
     2461 acaggcactt gccttcctgc atgtcgtgca gcttgatctt ctcgatcagg tacttggcgt
     2521 tctctttgcc ggtggtccgg atgatttcct cgatccgctc gttggtctgc cggttccgct
     2581 tctgcatctc gttgatcatt ttctgggcgt ccttggagtt cttctcgcgg gccagctcga
     2641 taatgatgtc gttgggcagg ccgtacttct tgatgatggc gttgatcact ttgatgctct
     2701 ggatgaagct tctcttcacg acggggctca ggatgaagtc gtccaccagg gtggtgggga
     2761 tctctttctg ctgggacagg tccaccttct tgggcaccag cttcagccgg ttgaagatag
     2821 cgatctggtt gtcgttggtg tgccacagct cgtccaggat caggttgatg gccttcaggc
     2881 tcaggttgtg ggtgccggta tagcccttca gattagagat ctgctcgatc tcttcctggg
     2941 tcagctcgga gttcagattg gtcagttctt cctggatgtc ctcgctgctc tggtagatgg
     3001 tcaggatctt ggcaatctga tccagcagct cggcgttctc aataatctct ttccgggcgg
     3061 taatgtcctt gatgtcgtgg tacaccttca ggttggtgaa ctcgggcttg ccggtgctgg
     3121 tcactctgta gcccttaata tcctcttcgt tcacgaggat ttctttggcg atctgcttca
     3181 gggtgggctt cttcttctgc ttgaacacgt tctcgatgat ctggaacttc tcgtaatatt
     3241 ccagcttctc gttctcgtcc ctggtgatca cgagattgtt caggtcgttc agggcgttgt
     3301 acaggtcggc gttgtaggcg tacttcacgc tccgcagttc ctcggggaag taggtgcagt
     3361 ggcccatcag catctcgtac cattctttga tgtccttcca gccgaagggg ctgccctcgc
     3421 caggtccctc atagtaggtc cgccgggttt ccagcaggtc gatgtaggtg tcgatgaagc
     3481 tctggtccag ctggtggtag gccttctgca ccttcagcag ctgtttggct tctttcacgt
     3541 agtcgctggt cttgaatctg ttgatgctgc cccgcacttc gccgtctttc ttcagccgtt
     3601 ccagctgcag ttcggccacg tatttctctt ccagggcctt gctgttccgg ctgatctgct
     3661 ctttggtgga cagctcgttg ccggtgtcct cttccacctc gttcacgttg tgcacgcctc
     3721 ttctcttggc caggtgcagc agggcggcag agaactcttc ctcgctcagc ttctggctca
     3781 ggcccttcac tctggcctcg taggggttga tgccgctcag ctcgctgtgg tcggtcagca
     3841 ggttgtagtc gaacagcagc ttcttcactc tctggattct atgccgcctc cgccgcttca
     3901 gccttctggc gcctctcttg ctccgcctgc cctcgttgtt ttccacgttg gcctctttga
     3961 acagccgcac gccggcatcg atcacgtccc gtgtctcgta gtcgatgatg ccgtagccca
     4021 cgctggtgat gccgatgtcc aggcccagga tgtagttccg cttggctgct gggactccgt
     4081 ggataccgac cttccgcttc ttctttgggg ccatgcggcc gctgcagacc ggtgctagca
     4141 taacttcgta taaagtatcc tatacgaagt tatttgcctt aacccagaaa ttatcactgt
     4201 tattctttag aatggtgcaa agaataactt cgtataatgt atgctatacg aagttatgaa
     4261 ttcctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     4321 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     4381 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     4441 tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct ggggaggtac
     4501 cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
     4561 gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
     4621 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     4681 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     4741 acgaaacacc ggacggcggc agagtctcat cgttttagta ctctggaaac agaatctact
     4801 aaaacaaggc aaaatgccgt gtttatctcg tcaacttgtt ggcgagattt ttgcggccgc
     4861 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     4921 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     4981 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     5041 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     5101 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
     5161 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     5221 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     5281 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     5341 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     5401 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
     5461 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     5521 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     5581 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     5641 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     5701 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     5761 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     5821 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     5881 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     5941 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     6001 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     6061 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     6121 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     6181 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     6241 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     6301 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     6361 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     6421 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     6481 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     6541 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     6601 tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg
     6661 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     6721 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     6781 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     6841 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     6901 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     6961 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     7021 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     7081 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     7141 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     7201 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     7261 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     7321 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     7381 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     7441 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     7501 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     7561 acggttcctg gccttttgct ggccttttgc tcacatgtcc tgcaggcagc tgcgcgctcg
     7621 ctcgctcact gaggccgccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg
     7681 agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcct
//
