LOCUS       sequence-307399-        4448 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  M7 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 4448)
  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 4448)
  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: pDGE501
FEATURES             Location/Qualifiers
     source          1..4448
                     /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        616..718
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             719..1402
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     complement(785..804)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1722..2027
                     /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     1906..1925
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(2227..2243)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2227..2243)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2240..2262)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2251..2267
                     /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(2275..2281,2282..2299,2300..2305))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2320..2341
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2458..2475)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2629..3217)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2709..2728)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3388..4179,4180..4248))
                     /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     4011..4030
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4249..4353)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4421..4439
                     /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 tggtctcacc agcaaaaact tcaaaatcta acacaaattt ttattgtatt
      481 tgagcagatt tgcataatag tcagcgaagc tgtgtcaagg gccaacatat tattcccaca
      541 tcggtaggtc aacacataaa ctccaggttt atgaggcttc gtagcagtag cacatgtcaa
      601 agtcttctga gacgttgatc ggcacgtaag aggttccaac tttcaccata atgaaataag
      661 atcactaccg ggcgtatttt ttgagttatc gagattttca ggagctaagg aagctaaaat
      721 ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca
      781 ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat
      841 tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca
      901 cattcttgcc cgcctgatga atgctcatcc ggaattccgt atggcaatga aagacggtga
      961 gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc aaactgaaac
     1021 gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac acatatattc
     1081 gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt ttattgagaa
     1141 tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc
     1201 caatatggac aacttcttcg cccccgtttt caccatgggc aaatattata cgcaaggcga
     1261 caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt
     1321 cggtagaatg cttaatgaat tacaacagta ctgcgatgag tggcaggggg ggcgtaaacg
     1381 ccgcgtggat ccggcttact aaaagccaga taacagtatg cgtatttgcg cgctgatttt
     1441 tgcggtataa gaatatatac tgatatgtat acccgaagta tgtcaaaaag aggtatgcta
     1501 tgaagcagcg tattacagtg acagttgaca gcgacagcta tcagttgctc aaggcatata
     1561 tgatgtcaat atctccggtc tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg
     1621 tgccgaacgc tggaaagcgg aaaatcagga agggatggct gaggtcgccc ggtttattga
     1681 aatgaacggc tcttttgctg acgagaacag gggctggtga aatgcagttt aaggtttaca
     1741 cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca
     1801 cgcccgggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct
     1861 cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg
     1921 atatggccag tgtgccggtt tccgttatcg gggaagaagt ggctgatctc agccaccgcg
     1981 aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggctccc
     2041 ttatacacag ccagtcgaag acttgtttca gagctatgct ggaaacagca tagcaagttg
     2101 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttttcg
     2161 aatgagacca tcggatcccg ggcccgtcga ctgcagaggc ctgcatgcaa gcttggcgta
     2221 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     2281 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     2341 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     2401 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     2461 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     2521 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     2581 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     2641 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     2701 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     2761 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     2821 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     2881 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     2941 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     3001 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     3061 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     3121 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     3181 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     3241 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     3301 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     3361 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     3421 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     3481 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag atccacgctc
     3541 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     3601 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     3661 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     3721 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     3781 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     3841 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     3901 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     3961 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     4021 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     4081 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     4141 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     4201 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     4261 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     4321 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     4381 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     4441 ctttcgtc
//
