LOCUS       sequence-382315-        5183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a gRNA that targets OXTR coding sequence in multiple 
            (rodent) species and eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5183)
  AUTHORS   Boender AJ, Boon M, Albers HE, Eck SR, Fricker BA, Kelly AM, LeDoux 
            JE, Motta SC, Shrestha P, Taylor JH, Trainor BC, Triana-Del Rio R, 
            Young LJ
  TITLE     An AAV-CRISPR/Cas9 strategy for gene editing across divergent rodent
            species: Targeting neural oxytocin receptors as a proof of concept.
  JOURNAL   Sci Adv. 2023 Jun 2;9(22):eadf4950. doi: 10.1126/sciadv.adf4950. 
            Epub 2023 May 31.
  PUBMED    37256960
REFERENCE   2  (bases 1 to 5183)
  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: "Sci Adv."; 
            date: "2023-06-2"; volume: "9(22):eadf4950. doi"; pages: " 
            10.1126/sciadv.adf4950. Epub 2023 May 31"
COMMENT     Sequence Label: pAAV-U6-sgRNA(OXTR.2)-CMV-eGFP
FEATURES             Location/Qualifiers
     source          1..5183
                     /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        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     145..165
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     316..335
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        414..489
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        583..886
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        887..1090
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1041..1061
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1129..1138
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1135..1137,1138..1140,1141..1851)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1135..1137,1138..1140,1141..1851)
                     /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(1180..1201)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1441..1460)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1788..1809
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1930..2406
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2129..2148)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2446..2586
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2446..2575
                     /label=AAV2 ITR
     rep_origin      2661..3116
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2748..2767)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2958..2979
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3133..3152)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3252..3274
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3312..3330)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3398..3502
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3503..3571,3572..4363)
                     /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(3721..3740)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4534..5122
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5023..5042
                     /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 gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accgtgatgt cccacagcag ctggttttag
      421 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      481 agtcggtgct ttttttctag agggtaccgg ggcccggtcg actagttatt aatagtaatc
      541 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      601 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      661 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      721 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      781 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      841 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
      901 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
      961 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     1021 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     1081 aagcagagct ggtttagtga accgtcagat ccgctagcgc taccggtcgc caccatggtg
     1141 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     1201 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     1261 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     1321 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     1381 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     1441 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     1501 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1561 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     1621 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     1681 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1741 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1801 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtccggactc
     1861 agatctcgat aacctgcagc gaattcgata tcaagcttat cgataccgag cgctgctcga
     1921 gagatctacg ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt
     1981 gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac
     2041 taggtgtcct tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt
     2101 gggaagacaa cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca
     2161 caatcttggc tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct
     2221 cccgagttgt tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg
     2281 tagagacggg gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc
     2341 tacccacctt ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct
     2401 gtccttctga ttttgtaggt aaccacgtgc ggaccgagcg gccgcaggaa cccctagtga
     2461 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     2521 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc
     2581 tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     2641 tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     2701 ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt
     2761 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     2821 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
     2881 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     2941 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc
     3001 gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga
     3061 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg
     3121 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     3181 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     3241 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     3301 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     3361 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3421 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3481 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     3541 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     3601 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     3661 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     3721 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     3781 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     3841 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     3901 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     3961 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     4021 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     4081 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     4141 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     4201 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     4261 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     4321 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     4381 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     4441 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     4501 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4561 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4621 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4681 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     4741 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     4801 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     4861 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     4921 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     4981 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5041 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5101 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5161 ttgctggcct tttgctcaca tgt
//
