LOCUS       sequence-196647-        2924 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For PCR amplification to create paired gRNAs to clone into Golden 
            Gate gRNA expression vectors (e.g. pSB700 Plasmid #64046).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2924)
  AUTHORS   Nageshwaran S, Chavez A, Cher Yeo N, Guo X, Lance-Byrne A, Tung A, 
            Collins JJ, Church GM
  TITLE     CRISPR Guide RNA Cloning for Mammalian Systems.
  JOURNAL   J Vis Exp. 2018 Oct 2;(140). doi: 10.3791/57998.
  PUBMED    30346403
REFERENCE   2  (bases 1 to 2924)
  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: "J Vis 
            Exp."; date: "2018-10-2"; volume: "(140). doi"; pages: " 
            10.3791/57998"
COMMENT     Sequence Label: pSN007
FEATURES             Location/Qualifiers
     source          1..2924
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    33..132
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(513..612)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(629..648)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(630..648)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(771..790)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             782..1591
                     /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="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(849..868)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1738..2326
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2227..2246
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2480..2497
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2656..2683)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2775..2861)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     join(2910..2924,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(2924,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aagctcgggc cccaaataat gattttattt tgactgatag
       61 tgacctgttc gttgcaacaa attgatgagc aatgcttttt tataatgcca actttgtaca
      121 aaaaagcagg ctacaaagtt ttagagctat gctgaaaagc atagcaagtt aaaataaggc
      181 agtgattttt aatccagtcc gtacacaact tgaaaaagtg cgcaccgatt cggtgctttt
      241 ttttcctacg cacctcgtcg aaccctcact gcagtattta gcatgcccca cccatctgca
      301 aggcattctg gatagtgtca aaacagccgg aaatcaagtc cgtttatctc aaactttagc
      361 attttgggaa taaatgatat ttgctatgct ggttaaatta gattttagtt aaatttcctg
      421 ctgaagctct agtacgataa gtaacttgac ctaagtgtaa agttgagatt tccttcaggt
      481 ttatatagct tgtgcgccgc ctgggtcacc gaacccagct ttcttgtaca aagttggcat
      541 tataagaaag cattgcttat caatttgttg caacgaacag gtcactatca gtcaaaataa
      601 aatcattatt tgccatccag ctgatatccc ctatagtgag tcgtattaca tggtcatagc
      661 tgtttcttgg cagctctggc ccgtgtctca aaatctctga tgttacattg cacaagataa
      721 aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
      781 tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga
      841 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
      901 cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
      961 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
     1021 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc
     1081 cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
     1141 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
     1201 cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga
     1261 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
     1321 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
     1381 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
     1441 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
     1501 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
     1561 gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg
     1621 gcagagcatt acgctgactt gacgggacgg cgcaagctca tgaccaaaat cccttaacgt
     1681 gagttacgcg tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     1741 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     1801 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     1861 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     1921 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     1981 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     2041 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     2101 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     2161 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     2221 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2281 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2341 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     2401 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     2461 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     2521 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     2581 cgactggaaa gcgggcagtg agcgcaacgc aattaatacg cgtaccgcta gccaggaaga
     2641 gtttgtagaa acgcaaaaag gccatccgtc aggatggcct tctgcttagt ttgatgcctg
     2701 gcagtttatg gcgggcgtcc tgcccgccac cctccgggcc gttgcttcac aacgttcaaa
     2761 tccgctcccg gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa
     2821 cgaaaggccc agtcttccga ctgagccttt cgttttattt gatgcctggc agttccctac
     2881 tctcgcgtta acgctagcat ggatgttttc ccagtcacga cgtt
//
