LOCUS       sequence-338867-        3513 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual targeted guide and cloning vector. Targets pEF-XMAS-2xStop. 
            Guides targeting endogenous sites can be cloned into BbsI sites..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3513)
  AUTHORS   Brookhouser N, Nguyen T, Tekel SJ, Standage-Beier K, Wang X, Brafman
            DA
  TITLE     A Cas9-mediated adenosine transient reporter enables enrichment of 
            ABE-targeted cells.
  JOURNAL   BMC Biol. 2020 Dec 14;18(1):193. doi: 10.1186/s12915-020-00929-7.
  PUBMED    33317513
REFERENCE   2  (bases 1 to 3513)
  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: "BMC Biol.";
            date: "2020-12-14"; volume: "18(1):193. doi"; pages: " 
            10.1186/s12915-020-00929-7"
COMMENT     Sequence Label: pDT-sgRNA-XMAS-2x
FEATURES             Location/Qualifiers
     source          1..3513
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        264..339
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(462..478)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(462..478)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(475..497)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    486..502
                     /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(510..516,517..534,535..540))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    555..576
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(693..710)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(864..1452)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(944..963)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1623..2414,2415..2483))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2246..2265
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2484..2588)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2656..2674
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2712..2734)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2834..2853
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3047..3069
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3061..3078
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3062..3078
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3085..3333
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        3354..3429
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        join(3510..3513,1..245)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
ORIGIN
        1 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
       61 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
      121 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
      181 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
      241 acaccgggtc ttcgagaaga cctgttttag agctagaaat agcaagttaa aataaggcta
      301 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgtttt agagctagaa
      361 atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg cgccaattct gcagacaaat
      421 ctagagtcga cctgcaggca tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg
      481 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
      541 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
      601 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
      661 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
      721 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
      781 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
      841 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
      901 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
      961 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     1021 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     1081 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     1141 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     1201 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     1261 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     1321 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     1381 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     1441 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     1501 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     1561 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     1621 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     1681 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     1741 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     1801 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     1861 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     1921 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     1981 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     2041 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     2101 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     2161 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     2221 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     2281 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     2341 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     2401 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     2461 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     2521 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     2581 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     2641 acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat
     2701 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     2761 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     2821 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa
     2881 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc attcaggctg
     2941 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
     3001 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt
     3061 tgtaaaacga cggccagtga attcgagggc ctatttccca tgattccttc atatttgcat
     3121 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
     3181 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
     3241 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
     3301 gctttatata tcttgtggaa aggacgaaac accgttgatg aggtggttca ggagttttag
     3361 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     3421 agtcggtgct tttttgtttt agagctagaa atagcaagtt aaaataaggc tagtccgttt
     3481 ttagcgcgtg cgccaattct gcagacagag agg
//
