LOCUS       sequence-307079-        4469 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  M1 shuttle vector containing the Solanum lycopersicum U3 promoter 
            for preparation of single guide RNA transcriptional units by cloning
            of hybridized oligonucleotides..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4469)
  AUTHORS   Stuttmann J, Barthel K, Martin P, Ordon J, Erickson JL, Herr R, 
            Ferik F, Kretschmer C, Berner T, Keilwagen J, Marillonnet S, Bonas U
  TITLE     Highly efficient multiplex editing: One-shot generation of 8x 
            Nicotiana benthamiana and 12x Arabidopsis mutants.
  JOURNAL   Plant J. 2021 Feb 12. doi: 10.1111/tpj.15197.
  PUBMED    33577114
REFERENCE   2  (bases 1 to 4469)
  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. 
            2021 Feb 12. doi: 10.1111/tpj.15197."
COMMENT     Sequence Label: pDGE474
FEATURES             Location/Qualifiers
     source          1..4469
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        635..737
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             738..1421
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     complement(804..823)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1741..2046
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1925..1944
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(2248..2264)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2248..2264)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2261..2283)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2272..2288
                     /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(2296..2302,2303..2320,2321..2326))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2341..2362
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2479..2496)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2650..3238)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2730..2749)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3409..4200,4201..4269))
                     /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     4032..4051
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4270..4374)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4442..4460
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa
      421 tgcatctaga tttggtctca ttgcgttttc ccagtcacga ccaaaaactt caaaatctaa
      481 cacaaatttt tattgtattt gagcagattt gcataatagt cagcgaagct gtgtcaaggg
      541 ccaacatatt attcccacat cggtaggtca acacataaac tccaggttta tgaggcttcg
      601 tagcagtagc acatgtcaaa gtcttctgag acgttgatcg gcacgtaaga ggttccaact
      661 ttcaccataa tgaaataaga tcactaccgg gcgtattttt tgagttatcg agattttcag
      721 gagctaagga agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc
      781 aatggcatcg taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc
      841 agaccgttca gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt
      901 tttatccggc ctttattcac attcttgccc gcctgatgaa tgctcatccg gaattccgta
      961 tggcaatgaa agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt
     1021 tccatgagca aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc
     1081 agtttctaca catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc
     1141 ctaaagggtt tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca
     1201 gttttgattt aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca
     1261 aatattatac gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg
     1321 tttgtgatgg cttccatgtc ggtagaatgc ttaatgaatt acaacagtac tgcgatgagt
     1381 ggcagggggg gcgtaaacgc cgcgtggatc cggcttacta aaagccagat aacagtatgc
     1441 gtatttgcgc gctgattttt gcggtataag aatatatact gatatgtata cccgaagtat
     1501 gtcaaaaaga ggtatgctat gaagcagcgt attacagtga cagttgacag cgacagctat
     1561 cagttgctca aggcatatat gatgtcaata tctccggtct ggtaagcaca accatgcaga
     1621 atgaagcccg tcgtctgcgt gccgaacgct ggaaagcgga aaatcaggaa gggatggctg
     1681 aggtcgcccg gtttattgaa atgaacggct cttttgctga cgagaacagg ggctggtgaa
     1741 atgcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta
     1801 cagagtgata ttattgacac gcccgggcga cggatggtga tccccctggc cagtgcacgt
     1861 ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc
     1921 tggcgcatga tgaccaccga tatggccagt gtgccggttt ccgttatcgg ggaagaagtg
     1981 gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga
     2041 atataaatgt caggctccct tatacacagc cagtcgaaga cttgtttcag agctatgctg
     2101 gaaacagcat agcaagttga aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     2161 agtcggtgct tttttttccc ttgagaccaa atcggatccc gggcccgtcg actgcagagg
     2221 cctgcatgca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     2281 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     2341 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     2401 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     2461 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     2521 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     2581 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     2641 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     2701 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     2761 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     2821 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     2881 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     2941 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     3001 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     3061 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     3121 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3181 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3241 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     3301 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     3361 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     3421 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     3481 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     3541 gataccgcga gatccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     3601 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     3661 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     3721 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     3781 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     3841 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     3901 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     3961 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     4021 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     4081 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     4141 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     4201 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     4261 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     4321 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     4381 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
     4441 aaataggcgt atcacgaggc cctttcgtc
//
