LOCUS       sequence-176427-        3620 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Subcloning of any sgRNA via BbsI sites. Note that there is an 
            improved version of this plasmid (Addgene 105866) that results in up
            to 10x greater efficiency.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3620)
  AUTHORS   Hahn F, Mantegazza O, Greiner A, Hegemann P, Eisenhut M, Weber AP
  TITLE     An Efficient Visual Screen for CRISPR/Cas9 Activity in Arabidopsis 
            thaliana.
  JOURNAL   Front Plant Sci. 2017 Jan 24;8:39. doi: 10.3389/fpls.2017.00039. 
            eCollection 2017.
  PUBMED    28174584
REFERENCE   2  (bases 1 to 3620)
  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: "Front Plant
            Sci."; date: "2017-01-24"; volume: "8:39. doi"; pages: " 
            10.3389/fpls.2017.00039. eCollection 2017"
COMMENT     Sequence Label: pFH6
FEATURES             Location/Qualifiers
     source          1..3620
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(44..62)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(45..62)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(80..96)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(80..96)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(93..115)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    104..120
                     /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(128..134,135..152,153..158))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    173..194
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(311..328)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(482..1070)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(562..581)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1241..2032,2033..2101))
                     /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     1864..1883
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2102..2206)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2284..2739)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2421..2442)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2633..2652
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2865..2887
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2879..2896
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2880..2896
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2903..2922
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2903..2921
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2952..2961
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2956..2965
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        3460..3535
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 atatgggaga gctcccaacg cgttggatgc atagcttgag tattctatag tgtcacctaa
       61 atagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
      121 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
      181 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
      241 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
      301 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
      361 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      421 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      481 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      541 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      601 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      661 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      721 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      781 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      841 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      901 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      961 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     1021 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     1081 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     1141 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     1201 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     1261 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1321 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1381 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1441 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1501 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1561 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1621 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1681 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1741 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1801 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1861 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1921 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1981 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     2041 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     2101 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     2161 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     2221 aagtgccacc tgatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     2281 aggaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     2341 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     2401 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     2461 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     2521 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     2581 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     2641 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     2701 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc cattcgccat tcaggctgcg
     2761 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
     2821 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
     2881 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gcccgacgtc
     2941 gcatgctccc ggccgccatg gcggccgcgg gaattcgatt cacgaagctt cgttgaacaa
     3001 cggaaactcg acttgccttc cgcacaatac atcatttctt cttagctttt tttcttcttc
     3061 ttcgttcata cagttttttt ttgtttatca gcttacattt tcttgaaccg tagctttcgt
     3121 tttcttcttt ttaactttcc attcggagtt tttgtatctt gtttcatagt ttgtcccagg
     3181 attagaatga ttaggcatcg aaccttcaag aatttgattg aataaaacat cttcattctt
     3241 aagatatgaa gataatcttc aaaaggcccc tgggaatctg aaagaagaga agcaggccca
     3301 tttatatggg aaagaacaat agtatttctt atataggccc atttaagttg aaaacaatct
     3361 tcaaaagtcc cacatcgctt agataagaaa acgaagctga gtttatatac agctagagtc
     3421 gaagtagtga ttgtcccttc ggggtcttcg agaagacctg ttttagagct agaaatagca
     3481 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcaaatt
     3541 tttttaaact agtggtgatc ttgctgaaaa actcgagcca tccggaagat cttgaattcg
     3601 cggccgcctg caggtcgacc
//
