LOCUS       Exported                4554 bp ds-DNA     circular SYN 29-APR-2025
DEFINITION  pAAV-U6sgRNA(SapI)_hSyn-GFP-KASH-bGH (SpGuide acceptor). AAV plasmid
            for sgRNA cloning. GFP-KASH fusion facilitates FACS sorting of cells
            and nuclei. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4554)
  AUTHORS   Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y, Trombetta J, Sur
            M, Zhang F
  TITLE     In vivo interrogation of gene function in the mammalian brain using 
            CRISPR-Cas9.
  JOURNAL   Nat Biotechnol. 2014 Oct 19. doi: 10.1038/nbt.3055.
  PUBMED    25326897
REFERENCE   2  (bases 1 to 4554)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Apr 29, 2025 from SnapGene 5.1.7
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2014 Oct 19. doi: 10.1038/nbt.3055."
FEATURES             Location/Qualifiers
     source          1..4554
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          993..1742
                     /organism="Mus musculus"
                     /chromosome="6"
                     /map="6 29.15 cM"
                     /strain="C57BL/6"
                     /mol_type="mRNA"
                     /note="color: #ffffff"
                     /db_xref="taxon:10090"
     repeat_region   1..141
                     /locus_tag="AAV2 ITR"
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #ff0078"
     repeat_region   12..141
                     /locus_tag="AAV2 ITR(1)"
                     /label=AAV2 ITR
                     /label=AAV2 ITR(1)
                     /note="color: #ff0078"
     misc_feature    158..398
                     /label=hU6
                     /note="color: #ffff00"
     misc_feature    408..427
                     /label=mrSnca
                     /note="color: #a6acb3"
     misc_feature    428..513
                     /label=Scaffold cr1
                     /note="color: #00ff00"
     misc_feature    520..833
                     /label=mU6
                     /note="color: #ff0000"
     protein_bind    714..747
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
                     /note="color: #31849b; direction: RIGHT"
     primer_bind     751..771
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
                     /note="color: black; sequence: CAGCACAAAAGGAAACTCACC"
     misc_feature    843..862
                     /locus_tag="DRSR2(1)"
                     /label=DRSR2
                     /label=DRSR2(1)
                     /note="color: #ff00ff"
     misc_feature    863..950
                     /label=Scaffold cr2
                     /note="color: #00ff00"
     misc_feature    963..985
                     /locus_tag="DRSR2(2)"
                     /label=DRSR2
                     /label=DRSR2(2)
                     /note="color: #ff00ff"
     CDS             993..1037
                     /codon_start=1
                     /label=Linker
                     /note="color: #ffff00"
                     /translation="GGGGSGGGGSGGGGS"
     misc_feature    1038..1043
                     /label=NcoI
                     /note="color: #a6acb3"
     CDS             1044..1742
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=oScarlet
                     /note="color: #ff0000; direction: RIGHT"
     primer_bind     1046..1062
                     /label=Green_Scarlet_F
                     /note="color: black; sequence: ggtgagcaagggcgagg; added: 
                     2023-01-13"
     primer_bind     complement(1177..1195)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
                     /note="color: black; sequence: TTGGTCACCTTCAGCTTGG"
     primer_bind     1445..1462
                     /label=mCherry-F
                     /note="mCherry, forward primer"
                     /note="color: black; sequence: CCCCGTAATGCAGAAGAAGA"
     misc_feature    1760..1774
                     /locus_tag="X"
                     /label=X
                     /note="color: #f8ff00"
     misc_feature    1781..1803
                     /locus_tag="DRSR2(1)"
                     /label=DRSR2
                     /label=DRSR2(1)
                     /note="color: #ff00ff"
     repeat_region   1817..1957
                     /locus_tag="AAV2 ITR(2)"
                     /label=AAV2 ITR
                     /label=AAV2 ITR(2)
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #ff0078"
     repeat_region   1817..1946
                     /locus_tag="AAV2 ITR(3)"
                     /label=AAV2 ITR
                     /label=AAV2 ITR(3)
                     /note="color: #ff0078"
     rep_origin      2032..2487
                     /direction=RIGHT
                     /locus_tag="f1 ori"
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; 
                     arrowindicates direction of (+) strand synthesis"
                     /note="color: #ffc0cb"
     primer_bind     complement(2119..2138)
                     /locus_tag="F1ori-R"
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
                     /note="color: #ffc0cb; direction: LEFT"
     primer_bind     2329..2350
                     /locus_tag="F1ori-F"
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
                     /note="color: #ffc0cb; direction: RIGHT"
     primer_bind     complement(2504..2523)
                     /locus_tag="pRS-marker"
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectablemarker"
                     /note="color: #ffc0cb; direction: LEFT"
     primer_bind     2623..2645
                     /locus_tag="pGEX 3'"
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
                     /note="color: #ffc0cb; direction: RIGHT"
     primer_bind     complement(2683..2701)
                     /locus_tag="pBRforEco"
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, 
                     forwardprimer"
                     /note="color: #ffc0cb; direction: LEFT"
     promoter        2769..2873
                     /gene="bla"
                     /locus_tag="AmpR promoter"
                     /label=AmpR promoter
                     /note="color: #ffc0cb"
     CDS             2874..3734
                     /codon_start=1
                     /gene="bla"
                     /locus_tag="AmpR"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin,and 
                     related antibiotics"
                     /note="color: #ffc0cb"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3092..3111)
                     /locus_tag="Amp-R"
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
                     /note="color: #ffc0cb; direction: LEFT"
     rep_origin      3905..4493
                     /direction=RIGHT
                     /locus_tag="ori"
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffc0cb"
     primer_bind     4394..4413
                     /locus_tag="pBR322ori-F"
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
                     /note="color: #ffc0cb; direction: RIGHT"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgct cacatgtgag ggcctatttc ccatgattcc
      181 ttcatatttg catatacgat acaaggctgt tagagagata attggaatta atttgactgt
      241 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
      301 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
      361 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgagg cttcaggctc
      421 atagtctgtt taagagctaa gctggaaaca gcatagcaag tttaaataag gctagtccgt
      481 tatcaacttg aaaaagtggc accgagtcgg tgcttttttg atccgacgcg ccatctctag
      541 gcccgcgccg gccccctcgc acggacttgt gggagaagct cggctactcc cctgccccgg
      601 ttaatttgca tataatattt cctagtaact atagaggctt aatgtgcgat aaaagacaga
      661 taatctgttc tttttaatac tagctacatt ttacatgata ggcttggatt tctataactt
      721 cgtatagcat acattatacg aagttataaa cagcacaaaa ggaaactcac cctaactgta
      781 aagtaattgt gtgttttgag actataagta tcccttggag aaccaccttg ttggaaacac
      841 cggcgatcgt aatcacccga gtgtttgaga gctaagcaga aagctgcata gcaagttcaa
      901 ataaggctag tccgtacaca acttgaaaaa gtggcagccg agtcggctgc ttttttctgc
      961 aggcgatcgt aatcacccga gtgggcgctc gaggtggagg cggttcaggc ggaggtggct
     1021 ctggcggtgg cggatcgcca tgggcggtga gcaagggcga ggcagtgatc aaggagttca
     1081 tgcggttcaa ggtgcacatg gagggctcca tgaacggcca cgagttcgag atcgagggcg
     1141 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg
     1201 gccccctgcc cttctcctgg gacatcctgt cccctcagtt catgtacggc tccagggcct
     1261 tcaccaagca ccccgccgac atccccgact actataagca gtccttcccc gagggcttca
     1321 agtgggaccg cgtgatgaac ttcgaggacg gcggcgccgt gaccgtgacc caggacacct
     1381 ccctggagga cggcaccctg atctacaagg tgaagctccg cggcaccaac ttccctcctg
     1441 acggccccgt aatgcagaag aaaacaatgg gctgggaagc gtccaccgag cggttgtacc
     1501 ccgaggacgg cgtgctgaag ggcgacatta agatggccct gcgcctgaag gacggcgggc
     1561 gctacctggc ggacttcaag accacctaca aggccaagaa gcccgtgcag atgcccggcg
     1621 cctacaacgt ggaccgcaag ttggacatca cctcccacaa cgaggactac accgtggtgg
     1681 aacagtacga acgctccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt
     1741 aatcgagaat tcgatatcat agctagctag ctagggatcc gcgatcgtaa tcacccgagt
     1801 gggctagagg ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     1861 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     1921 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     1981 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     2041 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     2101 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     2161 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     2221 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     2281 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     2341 tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta taagggattt
     2401 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     2461 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     2521 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     2581 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     2641 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     2701 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     2761 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     2821 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     2881 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     2941 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     3001 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     3061 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     3121 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     3181 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     3241 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     3301 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     3361 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     3421 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     3481 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     3541 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     3601 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     3661 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     3721 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     3781 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     3841 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3901 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3961 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     4021 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     4081 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     4141 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     4201 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     4261 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     4321 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     4381 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     4441 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     4501 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
