LOCUS       sequence-307143-        4473 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  M5 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 4473)
  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 4473)
  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: pDGE498
FEATURES             Location/Qualifiers
     source          1..4473
                     /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        639..741
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             742..1425
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     complement(808..827)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1745..2050
                     /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     1929..1948
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(2252..2268)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2252..2268)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2265..2287)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2276..2292
                     /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(2300..2306,2307..2324,2325..2330))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2345..2366
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2483..2500)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2654..3242)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2734..2753)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3413..4204,4205..4273))
                     /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     4036..4055
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4274..4378)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4446..4464
                     /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 gactgccagc ttctatgagt actgacaaaa acttcaaaat
      481 ctaacacaaa tttttattgt atttgagcag atttgcataa tagtcagcga agctgtgtca
      541 agggccaaca tattattccc acatcggtag gtcaacacat aaactccagg tttatgaggc
      601 ttcgtagcag tagcacatgt caaagtcttc tgagacgttg atcggcacgt aagaggttcc
      661 aactttcacc ataatgaaat aagatcacta ccgggcgtat tttttgagtt atcgagattt
      721 tcaggagcta aggaagctaa aatggagaaa aaaatcactg gatataccac cgttgatata
      781 tcccaatggc atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat
      841 aaccagaccg ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac
      901 aagttttatc cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaattc
      961 cgtatggcaa tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc
     1021 gttttccatg agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc
     1081 cggcagtttc tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat
     1141 ttccctaaag ggtttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc
     1201 accagttttg atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcaccatg
     1261 ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat
     1321 gccgtttgtg atggcttcca tgtcggtaga atgcttaatg aattacaaca gtactgcgat
     1381 gagtggcagg gggggcgtaa acgccgcgtg gatccggctt actaaaagcc agataacagt
     1441 atgcgtattt gcgcgctgat ttttgcggta taagaatata tactgatatg tatacccgaa
     1501 gtatgtcaaa aagaggtatg ctatgaagca gcgtattaca gtgacagttg acagcgacag
     1561 ctatcagttg ctcaaggcat atatgatgtc aatatctccg gtctggtaag cacaaccatg
     1621 cagaatgaag cccgtcgtct gcgtgccgaa cgctggaaag cggaaaatca ggaagggatg
     1681 gctgaggtcg cccggtttat tgaaatgaac ggctcttttg ctgacgagaa caggggctgg
     1741 tgaaatgcag tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga
     1801 tgtacagagt gatattattg acacgcccgg gcgacggatg gtgatccccc tggccagtgc
     1861 acgtctgctg tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga
     1921 aagctggcgc atgatgacca ccgatatggc cagtgtgccg gtttccgtta tcggggaaga
     1981 agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg
     2041 gggaatataa atgtcaggct cccttataca cagccagtcg aagacttgtt tcagagctat
     2101 gctggaaaca gcatagcaag ttgaaataag gctagtccgt tatcaacttg aaaaagtggc
     2161 accgagtcgg tgcttttttt ttgcgtgaga ccaaatcgga tcccgggccc gtcgactgca
     2221 gaggcctgca tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     2281 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     2341 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     2401 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     2461 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     2521 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     2581 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     2641 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     2701 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     2761 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2821 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     2881 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     2941 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3001 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3061 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     3121 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     3181 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     3241 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     3301 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     3361 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     3421 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     3481 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     3541 caatgatacc gcgagatcca cgctcaccgg ctccagattt atcagcaata aaccagccag
     3601 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     3661 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     3721 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     3781 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     3841 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     3901 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     3961 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4021 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4081 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     4141 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     4201 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     4261 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     4321 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     4381 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     4441 ataaaaatag gcgtatcacg aggccctttc gtc
//
