LOCUS       sequence-437145-        7187 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An adeno-associated viral vector expressing eGFP-KASH and two guides
            targeting mouse TACR1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7187)
  AUTHORS   Belilos A, Gray C, Sanders C, Black D, Mays E, Richie CT, Sengupta 
            A, Hake HS, Francis TC
  TITLE     Nucleus Accumbens Local Circuit for Cue-Dependent Aversive Learning.
  JOURNAL   bioRxiv [Preprint]. 2023 Oct 9:2023.02.06.527338. doi: 
            10.1101/2023.02.06.527338.
  PUBMED    36798245
REFERENCE   2  (bases 1 to 7187)
  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: "bioRxiv 
            [Preprint]. 2023 Oct 9:2023.02.06.527338. doi: 
            10.1101/2023.02.06.527338."
COMMENT     Sequence Label: pOTTC1642 - pAAV EF1a EGFP-KASH with mTACR1 gRNAs
FEATURES             Location/Qualifiers
     source          1..7187
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        156..469
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     388..408
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        492..567
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        684..1862
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          915..1853
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1810..1830
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1882..1891
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1888..1890,1891..1893,1894..2604)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1888..1890,1891..1893,1894..2604)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1933..1954)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2194..2213)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2541..2562
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2896..3484
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2949..2969)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3367..3378)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3516..3992
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3715..3734)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     misc_RNA        complement(4049..4124)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(4146..4394)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4204..4223)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4374..4394)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   4450..4590
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4450..4579
                     /label=AAV2 ITR
     rep_origin      4665..5120
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4752..4771)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4962..4983
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5137..5156)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5256..5278
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5316..5334)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5402..5506
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5507..5575,5576..6367)
                     /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(5725..5744)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6538..7126
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7027..7046
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtcgtctc agatagatcc gacgccgcca tctctaggcc
      181 cgcgccggcc ccctcgcaca gacttgtggg agaagctcgg ctactcccct gccccggtta
      241 atttgcatat aatatttcct agtaactata gaggcttaat gtgcgataaa agacagataa
      301 tctgttcttt ttaatactag ctacatttta catgataggc ttggatttct ataagagata
      361 caaatactaa attattattt taaaaaacag cacaaaagga aactcaccct aactgtaaag
      421 taattgtgtg ttttgagact ataaatatcc cttggagaaa agccttgttt gccgtatagg
      481 cggctgccca agttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
      541 cttgaaaaag tggcaccgag tcggtgcttt ttttgtactg agacgccgcg gtcgcgtaag
      601 ctttgcaaag atggataaag ttttaaacag agaggaatct ttgcagctaa tggaccttct
      661 aggtcttgaa aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc
      721 ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt
      781 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
      841 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
      901 ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt
      961 atggcccttg cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg
     1021 agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc
     1081 ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc
     1141 accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac
     1201 ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca
     1261 ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat
     1321 gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag
     1381 ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg
     1441 caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg
     1501 ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca
     1561 cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc
     1621 gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt
     1681 ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta
     1741 ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt
     1801 ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt
     1861 gaggtaccgg atccaccggt cgccaccatg gtgagcaagg gcgaggagct gttcaccggg
     1921 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     1981 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     2041 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     2101 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     2161 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     2221 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     2281 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     2341 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     2401 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     2461 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
     2521 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     2581 ctcggcatgg acgagctgta caagtccgga ctcagatctc gagaggagga ggaggagaca
     2641 gacagcagga tgccccacct cgacagcccc ggcagctccc agccgagacg ctccttcctc
     2701 tcaagggtga tcagggcagc gctaccgttg cagctgcttc tgctgctgct gctgctcctg
     2761 gcctgcctgc tacctgcctc tgaagatgac tacagctgca cccaggccaa caactttgcc
     2821 cgatccttct accccatgct gcggtacacc aacgggccac ctcccaccta ggaattcgat
     2881 atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     2941 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     3001 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     3061 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     3121 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     3181 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     3241 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
     3301 tttccttggc tgctcgccta tgttgccacc tggattctgc gcgggacgtc cttctgctac
     3361 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     3421 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     3481 ccgcatcgat accgagcgct gctcgagaga tctacgggtg gcatccctgt gacccctccc
     3541 cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt gtcctaataa
     3601 aattaagttg catcattttg tctgactagg tgtccttcta taatattatg gggtggaggg
     3661 gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc ggggtctatt
     3721 gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc gcctcctggg
     3781 ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca tgcatgacca
     3841 ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg gccaggctgg
     3901 tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg ctgggattac
     3961 aggcgtgaac cactgctccc ttccctgtcc ttctgatttt gtaggtaacc acaccgcggc
     4021 gtctcagtac gctcagcgga gaaaaaaagc accgactcgg tgccactttt tcaagttgat
     4081 aacggactag ccttatttta acttgctatt tctagctcta aaacctacgt tgcccaccac
     4141 ggaacggtgt ttcgtccttt ccacaagata tataaagcca agaaatcgaa atactttcaa
     4201 gttacggtaa gcatatgata gtccatttta aaacataatt ttaaaactgc aaactaccca
     4261 agaaattatt actttctacg tcacgtattt tgtactaata tctttgtgtt tacagtcaaa
     4321 ttaattccaa ttatctctct aacagccttg tatcgtatat gcaaatatga aggaatcatg
     4381 ggaaataggc cctcttcctg cccgaccttg gatcgatccg cctgtgagac gtgcggaccg
     4441 agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     4501 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     4561 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc
     4621 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     4681 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4741 cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4801 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     4861 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     4921 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     4981 tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     5041 ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     5101 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     5161 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     5221 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     5281 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     5341 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     5401 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5461 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     5521 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5581 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5641 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5701 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     5761 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     5821 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     5881 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     5941 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     6001 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     6061 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     6121 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     6181 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     6241 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     6301 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6361 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6421 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6481 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6541 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6601 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6661 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     6721 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     6781 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     6841 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     6901 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     6961 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     7021 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     7081 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     7141 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
