LOCUS       sequence-99924-e        3738 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway-Entry vector containing AtU6-26 promoter and sgRNA backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3738)
  AUTHORS   Fauser F, Schiml S, Puchta H
  TITLE     Both CRISPR/Cas-based nucleases and nickases can be used efficiently
            for genome engineering in Arabidopsis thaliana.
  JOURNAL   Plant J. 2014 Jul;79(2):348-59. doi: 10.1111/tpj.12554. Epub 2014 
            Jun 17.
  PUBMED    24836556
REFERENCE   2  (bases 1 to 3738)
  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"; doi: "10.1111/tpj.12554";
            journalName: "Plant J"; date: "2014-07"; volume: "79"; issue: "2"; 
            pages: "348-59"
COMMENT     Sequence Label: pEn-Chimera
FEATURES             Location/Qualifiers
     source          1..3738
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      33..42
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      37..46
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    61..160
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_RNA        567..642
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(654..678)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(684..783)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(863..881)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(864..881)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(899..915)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(899..915)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(912..934)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    923..939
                     /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(947..953,954..971,972..977))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    992..1013
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1130..1147)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1301..1889)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1381..1400)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2060..2851,2852..2920))
                     /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     2683..2702
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2921..3025)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3103..3558)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3240..3261)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3452..3471
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3684..3706
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3698..3715
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3699..3715
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(3722..3738,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(3722..3738,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat
       61 caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat tgatgagcaa
      121 tgctttttta taatgccaac tttgtacaaa aaagcaggct ctttttttct tcttcttcgt
      181 tcatacagtt tttttttgtt tatcagctta cattttcttg aaccgtagct ttcgttttct
      241 tctttttaac tttccattcg gagtttttgt atcttgtttc atagtttgtc ccaggattag
      301 aatgattagg catcgaacct tcaagaattt gattgaataa aacatcttca ttcttaagat
      361 atgaagataa tcttcaaaag gcccctggga atctgaaaga agagaagcag gcccatttat
      421 atgggaaaga acaatagtat ttcttatata ggcccattta agttgaaaac aatcttcaaa
      481 agtcccacat cgcttagata agaaaacgaa gctgagttta tatacagcta gagtcgaagt
      541 agtgattggg gtcttcgaga agacctgttt tagagctaga aatagcaagt taaaataagg
      601 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttc tagacccagc
      661 tttcttgtac aaagttggca ttaacccagc tttcttgtac aaagttggca ttataaaaaa
      721 taattgctca tcaatttgtt gcaacgaaca ggtcactatc agtcaaaata aaatcattat
      781 ttgatcacta gtgaattcgc ggccgcctgc aggtcgacca tatgggagag ctcccaacgc
      841 gttggatgca tagcttgagt attctatagt gtcacctaaa tagcttggcg taatcatggt
      901 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
      961 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     1021 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     1081 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     1141 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     1201 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     1261 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     1321 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     1381 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     1441 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     1501 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     1561 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     1621 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     1681 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     1741 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     1801 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     1861 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     1921 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     1981 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     2041 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     2101 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     2161 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     2221 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     2281 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     2341 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     2401 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     2461 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     2521 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     2581 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     2641 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     2701 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     2761 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     2821 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     2881 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     2941 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     3001 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gatgcggtgt
     3061 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta agcgttaata
     3121 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     3181 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     3241 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     3301 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     3361 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     3421 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     3481 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     3541 cgccgctaca gggcgcgtcc attcgccatt caggctgcgc aactgttggg aagggcgatc
     3601 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     3661 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt
     3721 gtaatacgac tcactata
//
