LOCUS       sequence-100126-        3766 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes CRISPR sgRNA for Gateway or conventional cloning of 1-3 
            sgRNAs into pDe-CAS9 or pCAS9-TPC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3766)
  AUTHORS   Schiml S, Fauser F, Puchta H
  TITLE     The CRISPR/Cas system can be used as nuclease for in planta gene 
            targeting and as paired nickases for directed mutagenesis in 
            Arabidopsis resulting in heritable progeny.
  JOURNAL   Plant J. 2014 Oct 18. doi: 10.1111/tpj.12704.
  PUBMED    25327456
REFERENCE   2  (bases 1 to 3766)
  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: "Plant J. 
            2014 Oct 18. doi: 10.1111/tpj.12704."
COMMENT     Sequence Label: pEn-C1.1
FEATURES             Location/Qualifiers
     source          1..3766
                     /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    62..161
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_RNA        581..656
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(668..692)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(711..810)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(891..909)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(892..909)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(927..943)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(927..943)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(940..962)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    951..967
                     /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(975..981,982..999,1000..1005))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1020..1041
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1158..1175)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1329..1917)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1409..1428)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2088..2879,2880..2948))
                     /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     2711..2730
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2949..3053)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3131..3586)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3268..3289)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3480..3499
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3712..3734
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3726..3743
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3727..3743
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(3750..3766,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(3750..3766,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat
       61 tcaaataatg attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgatgagca
      121 atgctttttt ataatgccaa ctttgtacaa aaaagcaggc tacgcgtcct gaggcttttt
      181 ttcttcttct tcgttcatac agtttttttt tgtttatcag cttacatttt cttgaaccgt
      241 agctttcgtt ttcttctttt taactttcca ttcggagttt ttgtatcttg tttcatagtt
      301 tgtcccagga ttagaatgat taggcatcga accttcaaga atttgattga ataaaacatc
      361 ttcattctta agatatgaag ataatcttca aaaggcccct gggaatctga aagaagagaa
      421 gcaggcccat ttatatggga aagaacaata gtatttctta tataggccca tttaagttga
      481 aaacaatctt caaaagtccc acatcgctta gataagaaaa cgaagctgag tttatataca
      541 gctagagtcg aagtagtgat tggggtcttc gagaagacct gttttagagc tagaaatagc
      601 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
      661 tttctagacc cagctttctt gtacaaagtt ggcattaacg cgtcctgagg acccagcttt
      721 cttgtacaaa gttggcatta taaaaaataa ttgctcatca atttgttgca acgaacaggt
      781 cactatcagt caaaataaaa tcattatttg aatcactagt gaattcgcgg ccgcctgcag
      841 gtcgaccata tgggagagct cccaacgcgt tggatgcata gcttgagtat tctatagtgt
      901 cacctaaata gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
      961 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     1021 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     1081 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     1141 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     1201 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     1261 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     1321 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     1381 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     1441 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     1501 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     1561 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     1621 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     1681 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     1741 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     1801 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     1861 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     1921 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     1981 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     2041 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     2101 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     2161 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     2221 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     2281 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     2341 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     2401 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     2461 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     2521 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     2581 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     2641 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     2701 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     2761 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     2821 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     2881 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     2941 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     3001 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     3061 ccccgaaaag tgccacctga tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     3121 ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     3181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     3241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     3301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     3361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     3421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     3481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     3541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat tcgccattca
     3601 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
     3661 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
     3721 gacgttgtaa aacgacggcc agtgaattgt aatacgactc actata
//
