LOCUS       sequence-148634-        4371 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA shuttle vector; module 7.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4371)
  AUTHORS   Ordon J, Gantner J, Kemna J, Schwalgun L, Reschke M, Streubel J, 
            Boch J, Stuttmann J
  TITLE     Generation of chromosomal deletions in dicotyledonous plants 
            employing a user-friendly genome editing toolkit.
  JOURNAL   Plant J. 2016 Aug 31. doi: 10.1111/tpj.13319.
  PUBMED    27579989
REFERENCE   2  (bases 1 to 4371)
  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. 
            2016 Aug 31. doi: 10.1111/tpj.13319."
COMMENT     Sequence Label: pDGE14
FEATURES             Location/Qualifiers
     source          1..4371
                     /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        550..652
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             653..1312
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(719..738)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1654..1959
                     /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     1838..1857
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        1997..2072
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2150..2166)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2150..2166)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2163..2185)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2174..2190
                     /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(2198..2204,2205..2222,2223..2228))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2243..2264
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2381..2398)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2552..3140)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2632..2651)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3311..4102,4103..4171))
                     /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     3934..3953
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4172..4276)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4344..4362
                     /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 ttggtctcac cagtaagttg aaaacaatct tcaaaagtcc cacatcgctt
      481 agataagaaa acgaagctga gtttatatac agctagagtc gaagtagtga ttgaagtctt
      541 ctgagacgtt gatcggcacg taagaggttc caactttcac cataatgaaa taagatcact
      601 accgggcgta ttttttgagt tatcgagatt ttcaggagct aaggaagcta aaatggagaa
      661 aaaaatcact ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga
      721 ggcatttcag tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc
      781 ctttttaaag accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct
      841 tgcccgcctg atgaatgctc atccggaatt ccgtatggca atgaaagacg gtgagctggt
      901 gatatgggat agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc
      961 atcgctctgg agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga
     1021 tgtggcgtgt tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt
     1081 tttcgtctca gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat
     1141 ggacaacttc ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt
     1201 gctgatgccg ctggcgattc aggttcatca tgccgtctgt gatggcttcc atgtcggcag
     1261 aatgcttaat gaattacaac agtactgcga tgagtggcag ggcggggcgt aatcgcgtgg
     1321 atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat
     1381 aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc tatgaagcag
     1441 cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca
     1501 atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac
     1561 gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg
     1621 gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa
     1681 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg
     1741 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa
     1801 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc
     1861 agtgtgccgg tttccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
     1921 atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc ccttatacac
     1981 agccagtcga agacttgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     2041 atcaacttga aaaagtggca ccgagtcggt gctttttttt cgaatgagac caatcggatc
     2101 ccgggcccgt cgactgcaga ggcctgcatg caagcttggc gtaatcatgg tcatagctgt
     2161 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     2221 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     2281 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     2341 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     2401 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     2461 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     2521 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2581 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     2641 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     2701 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     2761 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2821 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2881 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2941 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     3001 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3061 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3121 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     3181 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     3241 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3301 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3361 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3421 catctggccc cagtgctgca atgataccgc gagaaccacg ctcaccggct ccagatttat
     3481 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     3541 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     3601 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     3661 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     3721 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     3781 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     3841 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     3901 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     3961 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     4021 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     4081 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4141 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     4201 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4261 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     4321 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt c
//
