LOCUS       sequence-236004-        2757 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A plasmid for cloning Cre-dependent guide RNAs using a modified 
            mouse U6 promoter containing loxP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2757)
  AUTHORS   Back S, Necarsulmer J, Whitaker LR, Coke LM, Koivula P, Heathward 
            EJ, Fortuno LV, Zhang Y, Yeh CG, Baldwin HA, Spencer MD, 
            Mejias-Aponte CA, Pickel J, Hoffman AF, Spivak CE, Lupica CR, 
            Underhill SM, Amara SG, Domanskyi A, Anttila JE, Airavaara M, Hope 
            BT, Hamra FK, Richie CT, Harvey BK
  TITLE     Neuron-Specific Genome Modification in the Adult Rat Brain Using 
            CRISPR-Cas9 Transgenic Rats.
  JOURNAL   Neuron. 2019 Feb 8. pii: S0896-6273(19)30062-5. doi: 
            10.1016/j.neuron.2019.01.035.
  PUBMED    30792150
REFERENCE   2  (bases 1 to 2757)
  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: "Neuron. 
            2019 Feb 8. pii: S0896-6273(19)30062-5. doi: 
            10.1016/j.neuron.2019.01.035."
COMMENT     Sequence Label: pOTTC1181 - pmU6(loxP-STOP-loxP) BbsI gRNA
FEATURES             Location/Qualifiers
     source          1..2757
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1..17
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    complement(436..469)
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     protein_bind    complement(700..733)
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     misc_RNA        767..842
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1053..1070)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1053..1069)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      1110..1697
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1598..1617
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1709..1736)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(1760..2575)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCLKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2483..2502
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2576..2678)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(2679..2710)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
ORIGIN
        1 caggaaacag ctatgacatc aagctgacta gataatctag ctgatcgtgg accgatcata
       61 cgtataatgc cgtaagatca cgggtcgcag cacagctcgc ggtccagtag tgatcgacac
      121 tgctcgatcc gctcgcaccg gcgaattggg tacccgtctc agatagatcc gacgccgcca
      181 tctctaggcc cgcgccggcc ccctcgcaca gacttgtggg agaagctcgg ctactcccct
      241 gccccggtta atttgcatat aatatttcct agtaactata gaggcttaat gtgcgataaa
      301 agacagataa tctgttcttt ttaatactag ctacatttta catgataggc ttggatttct
      361 ataagagata caaatactaa attattattt taaaaaacag cacactcgag aactcaccct
      421 aactgtaaag ttagtataac ttcgtatagt ataaattata cgaagttata agccttgttt
      481 tttgcgattt ttgaattcgt tcctcagagg aactgacaag caccctaaca tcctattgga
      541 ggctcactca cgttttttct attttgtttc ttgacagcag agctcgttgc tcactgtata
      601 gctcaggttg gcctgacact gatgaggttc tccagtgact gcctctacct acctactggg
      661 atgacagagg tgtaccacca agccacgccc gggggatcca taacttcgta tagtataaat
      721 tatacgaagt tataagcctt gtttgtcttc gtgtacaaga agacctgttt tagagctaga
      781 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
      841 gctttttttg tactgagacg ccgcggtgga gctccagctt ttgtggatcg acgagagcag
      901 cgcgactgga tctgtcgccc gtctcaaacg caaccctccg gcggtcgcat atcattcagg
      961 acgagcctca gactccagcg taactggact gcaatcaact cactggctca ccttccggtc
     1021 cacgatcagc tagaatcaag ctgactagat aaactggccg tcgttttaca cgggtgggcc
     1081 tttcttcggt agaaaatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     1141 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     1201 ccaactcttt ttccgaggta actggcttca gcagagcgca gataccaaat actgttcttc
     1261 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     1321 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     1381 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     1441 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     1501 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     1561 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     1621 gtcctgtcgg gtttcgccac ctctgacttg agcatcgatt tttgtgatgc tcgtcagggg
     1681 ggcggagcct atggaaaaac gccagcaacg cagaaaggcc cacccgaagg tgagccaggt
     1741 gattacattt aggtcctcat tagaaaaact catcgagcat caagtgaaac tgcaatttat
     1801 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     1861 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     1921 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     1981 aatcaccatg agtgacgact gaatccggtg agaatggcaa gagtttatgc atttctttcc
     2041 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca ccaaccaaac
     2101 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgccg ttaaaaggac
     2161 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     2221 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttccct gggatcgcag
     2281 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     2341 taaattccgt cagccagttt agcctgacca tctcatctgt aacatcattg gcaacgctac
     2401 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     2461 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     2521 tgttggaatt taatcgcggc ttcaagcaag acgtttcccg ttgaatatgg ctcattttag
     2581 cttccttagc tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt
     2641 cattatggtg aaagttggaa cctcttacgt gccgatcaag tcaaaagcct ccggtcggag
     2701 gcttttgact ttctgctatg gaggtcaggt atgatttaaa tggtcagtat tgagcct
//
