LOCUS       sequence-307430-        3501 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intermediate sgRNA array cloning vector position 2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3501)
  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 3501)
  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: pDGE504
FEATURES             Location/Qualifiers
     source          1..3501
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     6..25
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     259..276
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        370..472
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             473..1156
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     complement(539..558)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1476..1781
                     /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     1660..1679
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(1862..1880)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1948..2052
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2053..2847
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSLREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      join(3018..3501,1..105)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
       61 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
      121 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
      181 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
      241 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
      301 aaaccgcctc tccccgcgcg ttggccgatt cattaatcac tctgtgaaga catccctttg
      361 ctgagacctt gatcggcacg taagaggttc caactttcac cataatgaaa taagatcact
      421 accgggcgta ttttttgagt tatcgagatt ttcaggagct aaggaagcta aaatggagaa
      481 aaaaatcact ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga
      541 ggcatttcag tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc
      601 ctttttaaag accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct
      661 tgcccgcctg atgaatgctc atccggaatt ccgtatggca atgaaagacg gtgagctggt
      721 gatatgggat agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc
      781 atcgctctgg agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga
      841 tgtggcgtgt tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt
      901 tttcgtttca gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat
      961 ggacaacttc ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt
     1021 gctgatgccg ctggcgattc aggttcatca tgccgtttgt gatggcttcc atgtcggcag
     1081 aatgcttaat gaattacaac agtactgcga tgagtggcag ggggggcgta aacgccgcgt
     1141 ggatccggct tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt
     1201 ataagaatat atactgatat gtatacccga agtatgtcaa aaagaggtat gctatgaagc
     1261 agcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt
     1321 caatatctcc ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga
     1381 acgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa
     1441 cggctctttt gctgacgaga acaggggctg gtgaaatgca gtttaaggtt tacacctata
     1501 aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcctg
     1561 ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg
     1621 aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg
     1681 ccagtgtgcc ggtttccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg
     1741 acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac
     1801 acagccagtc ggtctcagct tcggtttgtc ttcacgaagt ggctcttcag tggacgaaag
     1861 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     1921 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     1981 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     2041 aaaaggaaga gtatggctaa aatgagaata tcaccggaat tgaaaaaact gatcgaaaaa
     2101 taccgctgcg taaaagatac ggaaggaatg tctcctgcta aggtatataa gctggtggga
     2161 gaaaatgaaa acctatattt aaaaatgacg gacagccggt ataaagggac cacctatgat
     2221 gtggaacggg aaaaggacat gatgctatgg ctggaaggaa agctgcctgt tccaaaggtc
     2281 ctgcactttg aacggcatga tggctggagc aatctgctca tgagtgaggc cgatggcgtc
     2341 ctttgctcgg aagagtatga agatgaacaa agccctgaaa agattatcga gctgtatgcg
     2401 gagtgcatca ggctctttca ctccatcgac atatcggatt gtccctatac gaatagctta
     2461 gacagccgct tagccgaatt ggattactta ctgaataacg atctggccga tgtggattgc
     2521 gaaaactggg aagaggacac tccatttaaa gatccgcgcg agctgtatga ttttttaaag
     2581 acggaaaagc ccgaagagga acttgtcttt tcccacggcg acctgggaga cagcaacatc
     2641 tttgtgaaag atggcaaagt aagtggcttt attgatcttg ggagaagcgg cagggcggac
     2701 aagtggtatg acattgcctt ctgcgtccgg tcgctcaggg aggatatcgg ggaagaacag
     2761 tatgtcgagc tattttttga cttactgggg atcaagcctg attgggagaa aataaaatat
     2821 tatattttac tggatgaatt gttttagctg tcagaccaag tttactcata tatactttag
     2881 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     2941 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3001 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3061 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3121 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     3181 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3241 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3301 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3361 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     3421 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3481 ggagagcgca cgagggagct t
//
