LOCUS       sequence-382295-        5184 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a gRNA that targets the LacZ gene (serves as control) and 
            eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5184)
  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 5184)
  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(CTRL)-CMV-eGFP
FEATURES             Location/Qualifiers
     source          1..5184
                     /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        415..490
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        584..887
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        888..1091
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1042..1062
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1130..1139
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1136..1138,1139..1141,1142..1852)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1136..1138,1139..1141,1142..1852)
                     /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(1181..1202)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1442..1461)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1789..1810
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1931..2407
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2130..2149)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2447..2587
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2447..2576
                     /label=AAV2 ITR
     rep_origin      2662..3117
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2749..2768)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2959..2980
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3134..3153)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3253..3275
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3313..3331)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3399..3503
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3504..3572,3573..4364)
                     /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(3722..3741)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4535..5123
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5024..5043
                     /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 accggtgagc gagtaacaac ccgtgtttta
      421 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
      481 gagtcggtgc tttttttcta gagggtaccg gggcccggtc gactagttat taatagtaat
      541 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      601 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      661 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      721 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      781 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
      841 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
      901 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
      961 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     1021 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     1081 taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggtcg ccaccatggt
     1141 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1201 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     1261 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1321 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     1381 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     1441 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     1501 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1561 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     1621 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     1681 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     1741 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     1801 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtccggact
     1861 cagatctcga taacctgcag cgaattcgat atcaagctta tcgataccga gcgctgctcg
     1921 agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt
     1981 tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga
     2041 ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt
     2101 tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc
     2161 acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc
     2221 tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg
     2281 gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat
     2341 ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc
     2401 tgtccttctg attttgtagg taaccacgtg cggaccgagc ggccgcagga acccctagtg
     2461 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     2521 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     2581 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     2641 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2701 tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt
     2761 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2821 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     2881 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2941 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct
     3001 cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg
     3061 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     3121 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     3181 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     3241 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     3301 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     3361 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3421 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3481 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3541 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3601 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3661 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3721 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     3781 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3841 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3901 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3961 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     4021 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     4081 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     4141 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4201 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     4261 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4321 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4381 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4441 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4501 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4561 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4621 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4681 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4741 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4801 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4861 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4921 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4981 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5041 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5101 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5161 tttgctggcc ttttgctcac atgt
//
