LOCUS       sequence-201859-        2689 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Can be used as PCR template for a STAgR reaction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2689)
  AUTHORS   Breunig CT, Durovic T, Neuner AM, Baumann V, Wiesbeck MF, Koferle A,
            Gotz M, Ninkovic J, Stricker SH
  TITLE     One step generation of customizable gRNA vectors for multiplex 
            CRISPR approaches through string assembly gRNA cloning (STAgR).
  JOURNAL   PLoS One. 2018 Apr 27;13(4):e0196015. doi: 
            10.1371/journal.pone.0196015. eCollection 2018.
  PUBMED    29702666
REFERENCE   2  (bases 1 to 2689)
  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: "PLoS One.";
            date: "2018-04-27"; volume: "13(4):e0196015. doi"; pages: " 
            10.1371/journal.pone.0196015. eCollection 2018"
COMMENT     Sequence Label: STAGR_gRNAScaffold_mU6
FEATURES             Location/Qualifiers
     source          1..2689
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(474..1268)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(1269..1373)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1674..1696
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1688..1705
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1689..1705
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1716..1735
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1716..1734
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1739..1750
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     misc_RNA        1766..1841
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1861..2174
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     2093..2113
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     complement(2291..2308)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(2401..2689,1..300))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2481..2500)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
       61 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
      121 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
      181 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
      241 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
      301 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
      361 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      421 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttattagaaa
      481 aattcatcca gcagacgata aaacgcaata cgctggctat ccggtgccgc aatgccatac
      541 agcaccagaa aacgatccgc ccattcgccg cccagttctt ccgcaatatc acgggtggcc
      601 agcgcaatat cctgataacg atccgccacg cccagacggc cgcaatcaat aaagccgcta
      661 aaacggccat tttccaccat aatgttcggc aggcacgcat caccatgggt caccaccaga
      721 tcttcgccat ccggcatgct cgctttcaga cgcgcaaaca gctctgccgg tgccaggccc
      781 tgatgttctt catccagatc atcctgatcc accaggcccg cttccatacg ggtacgcgca
      841 cgttcaatac gatgtttcgc ctgatgatca aacggacagg tcgccgggtc cagggtatgc
      901 agacgacgca tggcatccgc cataatgctc actttttctg ccggcgccag atggctagac
      961 agcagatcct gacccggcac ttcgcccagc agcagccaat cacggcccgc ttcggtcacc
     1021 acatccagca ccgccgcaca cggaacaccg gtggtggcca gccagctcag acgcgccgct
     1081 tcatcctgca gctcgttcag cgcaccgctc agatcggttt tcacaaacag caccggacga
     1141 ccctgcgcgc tcagacgaaa caccgccgca tcagagcagc caatggtctg ctgcgcccaa
     1201 tcatagccaa acagacgttc cacccacgct gccgggctac ccgcatgcag gccatcctgt
     1261 tcaatcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     1321 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     1381 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     1441 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     1501 caaaagaata gaccgagata gggttgagtg gccgctacag ggcgctccca ttcgccattc
     1561 aggctgcgca actgttggga agggcgtttc ggtgcgggcc tcttcgctat tacgccagct
     1621 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
     1681 acgacgttgt aaaacgacgg ccagtgagcg cgacgtaata cgactcacta tagggcgaat
     1741 tgaaggaagg ccgtcaaggc cgcatgtttt agagctagaa atagcaagtt aaaataaggc
     1801 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttcg gtgctttttt
     1861 gatccgacgc cgccatctct aggcccgcgc cggccccctc gcacagactt gtgggagaag
     1921 ctcggctact cccctgcccc ggttaatttg catataatat ttcctagtaa ctatagaggc
     1981 ttaatgtgcg ataaaagaca gataatctgt tctttttaat actagctaca ttttacatga
     2041 taggcttgga tttctataag agatacaaat actaaattat tattttaaaa aacagcacaa
     2101 aaggaaactc accctaactg taaagtaatt gtgtgttttg agactataaa tatcccttgg
     2161 agaaaagcct tgtttgctgg gcctcatggg ccttcctttc actgcccgct ttccagtcgg
     2221 gaaacctgtc gtgccagctg cattaacatg gtcatagctg tttccttgcg tattgggcgc
     2281 tctccgcttc ctcgctcact gactcgctgc gctcggtcgt tcgggtaaag cctggggtgc
     2341 ctaatgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     2401 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     2461 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     2521 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     2581 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     2641 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcc
//
