LOCUS       sequence-305918-        4445 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  M4 shuttle vector containing the Solanum lycopersicum U6 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 4445)
  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 4445)
  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: pDGE659
FEATURES             Location/Qualifiers
     source          1..4445
                     /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        610..712
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             713..1396
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     complement(779..798)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1716..2021
                     /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     1900..1919
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(2224..2240)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2224..2240)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2237..2259)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2248..2264
                     /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(2272..2278,2279..2296,2297..2302))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2317..2338
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2455..2472)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2626..3214)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2706..2725)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3385..4176,4177..4245))
                     /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     4008..4027
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4246..4350)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4418..4436
                     /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 ttttggtctc aatggcaaga gaaataagaa aaaggccggc ccatttagat
      481 tgaagatcca ggcccaggcc cgtaaaagaa acccaacaag caaattctcc ctcatcgctt
      541 atacaaagct actttgcctc gtttatatag cggaatatga acatgtatga ttgaagtctt
      601 ctgagacgtt gatcggcacg taagaggttc caactttcac cataatgaaa taagatcact
      661 accgggcgta ttttttgagt tatcgagatt ttcaggagct aaggaagcta aaatggagaa
      721 aaaaatcact ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga
      781 ggcatttcag tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc
      841 ctttttaaag accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct
      901 tgcccgcctg atgaatgctc atccggaatt ccgtatggca atgaaagacg gtgagctggt
      961 gatatgggat agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc
     1021 atcgctctgg agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga
     1081 tgtggcgtgt tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt
     1141 tttcgtctca gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat
     1201 ggacaacttc ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt
     1261 gctgatgccg ctggcgattc aggttcatca tgccgtttgt gatggcttcc atgtcggcag
     1321 aatgcttaat gaattacaac agtactgcga tgagtggcag ggggggcgta aacgccgcgt
     1381 ggatccggct tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt
     1441 ataagaatat atactgatat gtatacccga agtatgtcaa aaagaggtat gctatgaagc
     1501 agcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt
     1561 caatatctcc ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga
     1621 acgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa
     1681 cggctctttt gctgacgaga acaggggctg gtgaaatgca gtttaaggtt tacacctata
     1741 aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg
     1801 ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg
     1861 aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg
     1921 ccagtgtgcc ggtttccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg
     1981 acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac
     2041 acagccagtc gaagacttgt ttcagagcta tgctggaaac agcatagcaa gttgaaataa
     2101 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt ttgacttgag
     2161 accaaaatcg gatcccgggc ccgtcgactg cagaggcctg catgcaagct tggcgtaatc
     2221 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     2281 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     2341 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     2401 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     2461 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     2521 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     2581 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     2641 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     2701 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     2761 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     2821 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     2881 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     2941 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3001 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3061 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3121 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3181 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3241 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3301 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3361 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     3421 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     3481 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagatc cacgctcacc
     3541 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     3601 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     3661 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     3721 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     3781 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     3841 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     3901 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     3961 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     4021 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     4081 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     4141 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     4201 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4261 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4321 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     4381 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     4441 tcgtc
//
