LOCUS       sequence-338865-        3512 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual targeted guide and cloning vector. Targets pEF-XMAS-1xStop. 
            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 3512)
  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 3512)
  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-1x
FEATURES             Location/Qualifiers
     source          1..3512
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        54..129
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        210..458
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        477..552
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(674..690)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(674..690)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(687..709)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    698..714
                     /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(722..728,729..746,747..752))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    767..788
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(905..922)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1076..1664)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1156..1175)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1835..2626,2627..2695))
                     /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     2458..2477
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2696..2800)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2868..2886
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2924..2946)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3046..3065
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3259..3281
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3273..3290
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3274..3290
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        join(3297..3512,1..33)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
ORIGIN
        1 gctttatata tcttgtggaa aggacgaaac accgttgatg gggtggttca ggagttttag
       61 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      121 agtcggtgct tttttgtttt agagctagaa atagcaagtt aaaataaggc tagtccgttt
      181 ttagcgcgtg cgccaattct gcagacagag agggcctatt tcccatgatt ccttcatatt
      241 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
      301 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
      361 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
      421 tcttggcttt atatatcttg tggaaaggac gaaacaccgg gtcttcgaga agacctgttt
      481 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
      541 ccgagtcggt gcttttttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
      601 tttttagcgc gtgcgccaat tctgcagcaa atctagagtc gacctgcagg catgcaagct
      661 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
      721 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
      781 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
      841 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
      901 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
      961 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     1021 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     1081 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     1141 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     1201 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     1261 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     1321 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     1381 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     1441 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     1501 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     1561 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     1621 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     1681 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     1741 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     1801 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     1861 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     1921 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     1981 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     2041 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     2101 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     2161 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     2221 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     2281 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     2341 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     2401 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     2461 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     2521 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     2581 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     2641 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     2701 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     2761 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     2821 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     2881 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     2941 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     3001 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     3061 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     3121 accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     3181 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
     3241 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattcgagg
     3301 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     3361 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     3421 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     3481 ttaccgtaac ttgaaagtat ttcgatttct tg
//
