LOCUS       sequence-307181-        4450 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  M6E 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 4450)
  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 4450)
  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: pDGE500
FEATURES             Location/Qualifiers
     source          1..4450
                     /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"
     CDS             complement(576..881)
                     /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     complement(678..697)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1201..1884)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     1799..1818
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1885..1987)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2229..2245)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2229..2245)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2242..2264)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2253..2269
                     /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(2277..2283,2284..2301,2302..2307))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2322..2343
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2460..2477)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2631..3219)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2711..2730)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3390..4181,4182..4250))
                     /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     4013..4032
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4251..4355)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4423..4441
                     /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 taaggtctct aagcaaaaaa aagcaccgac tcggtgccac tttttcaagt
      481 tgataacgga ctagccttat ttcaacttgc tatgctgttt ccagcatagc tctgaaacaa
      541 gtcttcgact ggctgtgtat aagggagcct gacatttata ttccccagaa catcaggtta
      601 atggcgtttt tgatgtcatt ttcgcggtgg ctgagatcag ccacttcttc cccgataacg
      661 gaaaccggca cactggccat atcggtggtc atcatgcgcc agctttcatc cccgatatgc
      721 accaccgggt aaagttcacg ggagacttta tctgacagca gacgtgcact ggccaggggg
      781 atcaccatcc gtcgcccggg cgtgtcaata atatcactct gtacatccac aaacagacga
      841 taacggctct ctcttttata ggtgtaaacc ttaaactgca tttcaccagc ccctgttctc
      901 gtcagcaaaa gagccgttca tttcaataaa ccgggcgacc tcagccatcc cttcctgatt
      961 ttccgctttc cagcgttcgg cacgcagacg acgggcttca ttctgcatgg ttgtgcttac
     1021 cagaccggag atattgacat catatatgcc ttgagcaact gatagctgtc gctgtcaact
     1081 gtcactgtaa tacgctgctt catagcatac ctctttttga catacttcgg gtatacatat
     1141 cagtatatat tcttataccg caaaaatcag cgcgcaaata cgcatactgt tatctggctt
     1201 ttagtaagcc ggatccacgc ggcgtttacg ccccccctgc cactcatcgc agtactgttg
     1261 taattcatta agcattctac cgacatggaa gccatcacaa acggcatgat gaacctgaat
     1321 cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg
     1381 ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg
     1441 attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc
     1501 accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta
     1561 ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg
     1621 aacactatcc catatcacca gctcaccgtc tttcattgcc atacggaatt ccggatgagc
     1681 attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt
     1741 tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc
     1801 aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt
     1861 atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc tcgataactc
     1921 aaaaaatacg cccggtagtg atcttatttc attatggtga aagttggaac ctcttacgtg
     1981 ccgatcaacg tctcagaaga ctttgacatg tgctactgct acgaagcctc ataaacctgg
     2041 agtttatgtg ttgacctacc gatgtgggaa taatatgttg gcccttgaca cagcttcgct
     2101 gactattatg caaatctgct caaatacaat aaaaatttgt gttagatttt gaagtttttg
     2161 cgcatgagac catcggatcc cgggcccgtc gactgcagag gcctgcatgc aagcttggcg
     2221 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     2281 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     2341 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     2401 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     2461 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     2521 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     2581 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     2641 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     2701 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     2761 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     2821 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     2881 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     2941 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     3001 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     3061 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     3121 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     3181 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     3241 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     3301 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     3361 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     3421 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     3481 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agatccacgc
     3541 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     3601 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     3661 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     3721 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     3781 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     3841 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     3901 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     3961 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     4021 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     4081 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     4141 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     4201 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     4261 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     4321 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     4381 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     4441 ccctttcgtc
//
