LOCUS       sequence-251645-        3193 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Embty backbone vector for expression of gRNA for spCas9 genome 
            editing or epigenome editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3193)
  TITLE     Expression vector for gRNA
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3193)
  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: 
            "Unpublished"
COMMENT     Sequence Label: gRNA_Cloning Vector BbsI
FEATURES             Location/Qualifiers
     source          1..3193
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     405..424
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        405..423
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        543..791
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     543..563
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     714..733
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        810..885
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(897..921)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(982..1000)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(983..1000)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1018..1034)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1018..1034)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1031..1053)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1042..1058
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1066..1072,1073..1090,1091..1096))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1111..1132
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1249..1266)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1420..2008)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1500..1519)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2166..2981)
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTSAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2889..2908
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2973..2992
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     3166..3184
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaatattac tacaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgagct agtgtaatac gactcactat
      421 agggcgcggc cgcagaattc gagctcggta cccgggatct cgaggccaga tcttgtacaa
      481 aaaagcaggc tttaaaggaa ccaattcagt cgactggatc cggtaccaag gtcgggcagg
      541 aagagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
      601 agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
      661 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
      721 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
      781 gacgaaacac cgggtcttcg agaagacctg ttttagagct agaaatagca agttaaaata
      841 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttctagaccc
      901 agctttcttg tacaaagttg gcattaattg tggatccgct ctagagtcga cctgcaggca
      961 tgcaagcttg cggccgcgta ttctatagtg tcacctaaat agcatggcgt aatcatggtc
     1021 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     1081 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     1141 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     1201 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     1261 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     1321 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     1381 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     1441 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     1501 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     1561 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     1621 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     1681 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     1741 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     1801 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     1861 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     1921 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     1981 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     2041 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     2101 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatgt
     2161 tttaattaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     2221 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     2281 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     2341 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     2401 acgactgaat ccggtgagaa tggcaatagc ttatgcattt ctttccagac ttgttcaaca
     2461 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     2521 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     2581 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     2641 ggatattctt ctaatacctg gaatgctgtt ttccctggga tcgcagaggt gagtaaccat
     2701 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     2761 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     2821 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     2881 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     2941 cgcggccttg agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     3001 tttatgtaag cagacataag catttatcag ggttattgtc tcatgagcgg atacatattt
     3061 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     3121 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     3181 aggccctttc gtc
//
