LOCUS       pHnS_KI;Actb_Donor;3xV5_KO;Dlg4        4812 bp    DNA     circular UNA 11-JUL-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   urn.local...1w-jeblee4
VERSION     urn.local...1w-jeblee4
KEYWORDS    .
SOURCE      
  ORGANISM  .
FEATURES             Location/Qualifiers
     source          1..4812
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /note="inverted terminal repeat of adeno-associated virus
                     serotype 2"
                     /standard_name="AAV2 ITR"
     repeat_region   12..141
                     /standard_name="AAV2 ITR"
     misc_feature    153..171
                     /standard_name="Primer site #1"
     misc_feature    172..461
                     /standard_name="bU6"
     misc_feature    462..481
                     /standard_name=1
     misc_feature    464..469
                     /standard_name="BbsI"
     misc_feature    482..567
                     /standard_name="Scaffold cr1"
     misc_feature    568..574
                     /standard_name="polyT"
     misc_feature    575..594
                     /standard_name="Primer site #2"
     misc_feature    595..908
                     /standard_name="mU6"
     protein_bind    789..822
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core
                     sequence (GCATACAT)."
                     /standard_name="loxP"
     primer_bind     826..846
                     /standard_name="mU6-F"
     primer_bind     complement(887..908)
                     /standard_name="SEQ_mU6_last_R"
     misc_feature    909..928
                     /standard_name=2
     misc_feature    929..1016
                     /standard_name="Scaffold cr2"
     misc_feature    1017..1023
                     /standard_name="polyT"
     misc_feature    1024..1042
                     /standard_name="Primer site #3"
     source          1043..1052
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1053..1319
                     /standard_name="hU6"
     primer_bind     1071..1091
                     /standard_name="hU6-F"
     misc_feature    1320..1339
                     /standard_name=3
     misc_feature    1332..1337
                     /standard_name="BbsI"
     misc_feature    1340..1427
                     /standard_name="Scaffold cr3"
     misc_feature    1428..1434
                     /standard_name="polyT"
     misc_feature    1435..1456
                     /standard_name="Primer site #4"
     regulatory      1466..1680
                     /note="Geneious type: promoter"
                     /note="human H1 RNA promoter"
                     /standard_name="H1 promoter"
     misc_feature    1487..2073
                     /standard_name="TwistBio"
     primer_bind     1601..1620
                     /standard_name="H1"
     misc_feature    1686..1705
                     /standard_name="mrActb"
     misc_feature    1706..1793
                     /standard_name="Scaffold cr4"
     misc_feature    1794..1800
                     /standard_name="polyT"
     misc_feature    complement(1817..1839)
                     /standard_name="mrActb"
     CDS             complement(1848..1889)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /transl_table=1
                     /translation="GKPIPNPLLGLDST"
                     /standard_name="V5 tag"
     CDS             complement(1899..1940)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /transl_table=1
                     /translation="GKPIPNPLLGLDST"
                     /standard_name="V5 tag"
     CDS             complement(1950..1991)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /transl_table=1
                     /translation="GKPIPNPLLGLDST"
                     /standard_name="V5 tag"
     CDS             complement(1992..1994)
                     /codon_start=1
                     /transl_table=1
                     /translation="M"
                     /standard_name="M"
     misc_feature    1995..2000
                     /standard_name="Kozak"
     source          2007..2021
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    2007..2021
                     /locus_tag="X"
                     /standard_name="X"
     source          2022..2023
                     /chromosome=5
                     /db_xref="taxon:10090"
                     /map="5 81.8 cM"
                     /mol_type="mRNA"
                     /organism="Mus musculus"
     misc_feature    complement(2024..2046)
                     /standard_name="mrActb"
     repeat_region   2075..2215
                     /note="inverted terminal repeat of adeno-associated virus
                     serotype 2"
                     /standard_name="AAV2 ITR"
     repeat_region   2075..2204
                     /standard_name="AAV2 ITR"
     rep_origin      2290..2745
     primer_bind     complement(2377..2396)
                     /standard_name="F1ori-R"
     primer_bind     2587..2608
                     /standard_name="F1ori-F"
     primer_bind     complement(2762..2781)
                     /standard_name="pRS-marker"
     primer_bind     2881..2903
                     /standard_name="pGEX 3'"
     primer_bind     complement(2941..2959)
                     /standard_name="pBRforEco"
     regulatory      3027..3131
                     /note="Geneious type: promoter"
                     /gene="bla"
                     /standard_name="AmpR promoter"
     CDS             join(3132..3200,3201..3992)
                     /codon_start=1
                     /gene="bla"
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /product="beta-lactamase"
                     /transl_table=1
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW*"
                     /standard_name="AmpR"
     primer_bind     complement(3350..3369)
                     /standard_name="Amp-R"
     rep_origin      4163..4751
     primer_bind     4652..4671
                     /standard_name="pBR322ori-F"
ORIGIN      
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgcc ggtaggtccc tcgacgttaa ccgtgaccga
      181 gcttgtctgc ccgcgcagtc cactagacag acgcgcagag gtcgggcggg cctagggcca
      241 atttcccatg gttcccttca tttgcatata tgatgtaata aggttatgga gactattaaa
      301 cttagcccta atcaaactat atgatgataa tgtgtgtggt acaaaaggtc ataacttatt
      361 atatactttg aaacttaaaa aagggttaca gtttagtcac cataactgta aaattttttc
      421 tattcttagc tttatatagt tcttgagagg ccatgtttat gaggcggatc gtgatccatc
      481 ggtttaagag ctaagctgga aacagcatag caagtttaaa taaggctagt ccgttatcaa
      541 cttgaaaaag tggcaccgag tcggtgcttt ttttccagtc tgactatcac gatggatccg
      601 acgcgccatc tctaggcccg cgccggcccc ctcgcacgga cttgtgggag aagctcggct
      661 actcccctgc cccggttaat ttgcatataa tatttcctag taactataga ggcttaatgt
      721 gcgataaaag acagataatc tgttcttttt aatactagct acattttaca tgataggctt
      781 ggatttctat aacttcgtat agcatacatt atacgaagtt ataaacagca caaaaggaaa
      841 ctcaccctaa ctgtaaagta attgtgtgtt ttgagactat aagtatccct tggagaacca
      901 ccttgttgcc gcaatgccag tcacgaacgt ttgagagcta agcagaaagc tgcatagcaa
      961 gttcaaataa ggctagtccg tacacaactt gaaaaagtgg cagccgagtc ggctgctttt
     1021 tttgctcgaa tctacactca gctatggcgc gccccaaggt cgggcaggaa gagggcctat
     1081 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
     1141 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
     1201 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
     1261 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagcc agaaacatgc
     1321 gtcatgcacg aggatgccgg tttcagagct aagcacaaga gtgcatagca agttgaaata
     1381 aggctagtcc gtttacaact tgaaaaagtg gcacccgagt cgggtgcttt ttttctcgag
     1441 tactaggatc cattaggcgg tctaggaacg ctgacgtcat caacccgctc caaggaatcg
     1501 cgggcccagt gtcactaggc gggaacaccc agcgcgcgtg cgccctggca ggaagatggc
     1561 tgtgagggac aggggagtgg cgccctgcaa tatttgcatg tcgctatgtg ttctgggaaa
     1621 tcaccataaa cgtgaaatgt ctttggattt gggaatctta taagttctgt atgagaccac
     1681 caccgcgcag cgatatcgtc atccagttta agagctaagc tcagaagagc atagcaagtt
     1741 taaataaggc tagtccgtac tcaacttgaa aaagtggcag ccgagtcggc tgcttttttt
     1801 ggtctgagac gccgcgccat ggatgacgat atcgctgcgg tgatccccgt ggagtccagg
     1861 cccaacagag ggttagggat aggcttacct tgttgctgtg tgctatccag accgagaagt
     1921 gggtttggaa tgggcttacc ctgctgctga gtactatcaa gtccaaggag ggggttcggt
     1981 ataggttttc ccatggtggc ctcgagtagc tagctagcta gccccatgga tgacgatatc
     2041 gctgcgcacg cgtgtgtcta gactgcgcgg ccgcaggaac ccctagtgat ggagttggcc
     2101 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     2161 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc
     2221 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     2281 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2341 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2401 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2461 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     2521 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2581 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg ggctattctt
     2641 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     2701 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     2761 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     2821 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     2881 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     2941 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     3001 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3061 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3121 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     3181 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     3241 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     3301 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     3361 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     3421 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     3481 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     3541 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     3601 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     3661 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     3721 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     3781 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     3841 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     3901 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     3961 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     4021 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     4081 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4141 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4201 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4261 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     4321 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4381 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4441 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4501 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4561 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     4621 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     4681 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     4741 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4801 ttgctcacat gt
//
