LOCUS       sequence-331272-        3371 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A Golden Gate (MoClo) compatible Level 2 acceptor binary vector 
            based on pGreenII (Hellens et al., 2000)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3371)
  AUTHORS   Smedley MA, Hayta S, Clarke M, Harwood WA
  TITLE     CRISPR-Cas9 Based Genome Editing in Wheat.
  JOURNAL   Curr Protoc. 2021 Mar;1(3):e65. doi: 10.1002/cpz1.65.
  PUBMED    33687760
REFERENCE   2  (bases 1 to 3371)
  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: "Curr 
            Protoc."; date: "2021-03"; volume: "1(3):e65. doi"; pages: " 
            10.1002/cpz1.65"
COMMENT     Sequence Label: pGoldenGreenGate-M (pGGG-M)
FEATURES             Location/Qualifiers
     source          1..3371
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(80..668)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(160..179)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(839..1654)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(839..1654)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1562..1581
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1646..1665
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1945..2380
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     misc_feature    2507..2529
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     misc_feature    2613..2637
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     protein_bind    2782..2803
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2818..2823,2824..2841,2842..2848)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2856..2872
                     /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)."
     primer_bind     2861..2883
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2880..2896
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2880..2896
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2892..3215
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    2909..2965
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(2966..2983)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2966..2982)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2975..2997)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3191..3210)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    3336..3360
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
ORIGIN
        1 atgtgtttgt ttgagatctc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
       61 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
      121 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
      181 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
      241 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
      301 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
      361 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
      421 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
      481 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
      541 ctctgctgaa gccagttacc ttcggaagaa gagttggtag ctcttgatcc ggcaaacaaa
      601 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
      661 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
      721 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
      781 attaaaaatg aagttttaaa tcaatctaaa gtatatatgt gtaacattgg tctagtgatt
      841 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
      901 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
      961 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     1021 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     1081 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
     1141 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     1201 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     1261 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     1321 cttctaatac ctggaatgct gttttccctg ggatcgcagt ggtgagtaac catgcatcat
     1381 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     1441 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     1501 actctggcgc atcgggcttc ccatacaatc ggtagattgt cgcacctgat tgcccgacat
     1561 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     1621 ttgagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     1681 aagcagacag ttttattgtt catgatgata tatttttatc ttgtgcaatg taacatcaga
     1741 gattttgaga cacaacgtgg ctttgttgaa taaatcgaac ttttgctgag ttgaaggatc
     1801 agatcacgca tcttcccgac aacgcagacc gttccgtggc aaagcaaaag ttcaaaatcc
     1861 ccaactggtc cacctacaac aaagctctca tcaaccgtgg ctccctcact ttctggctgg
     1921 atgatggggc gattcaggcg atccccatcc aacagcccgc cgtcgagcgg gcttttttat
     1981 ccccggaagc ctgtggatag agggtagtta tccacgtgaa accgctaatg ccccgcaaag
     2041 ccttgattca cggggctttc cggcccgctc caaaaactat ccacgtgaaa tcgctaatca
     2101 gggtacgtga aatcgctaat cggagtacgt gaaatcgcta ataaggtcac gtgaaatcgc
     2161 taatcaaaaa ggcacgtgag aacgctaata gccctttcag atcaacagct tgcaaacacc
     2221 cctcgctccg gcaagtagtt acagcaagta gtatgttcaa ttagcttttc aattatgaat
     2281 atatatatca attattggtc gcccttggct tgtggacaat gcgctacgcg caccggctcc
     2341 gcccgtggac aaccgcaagc ggttgcccac cgtcgagcgc ctttgcccac aacccggcgg
     2401 ccggccgcaa cagatcgttt tataaatttt tttttttgaa aaagaaaaag cccgaaaggc
     2461 ggcaacctct cgggcttctg gatttccgat ccccggaatt agatcttggc aggatatatt
     2521 gtggtgtaac gtttagtcat ggttgatggg ctgcctgtat cgagtggtga ttttgtgccg
     2581 agctgccggt cggggagctg ttggctggct ggtggcagga tatattgtgg tgtaaacaaa
     2641 ttgacgctta gacaacttaa taacacattg cggacgtttt taatgtactg gggttgaaca
     2701 ctctgtgggt ctcatgcctt gtcttcgcag ctggcacgac aggtttcccg actggaaagc
     2761 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     2821 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     2881 aggaaacagc tatgaccatg attacgccaa gcttgcatgc ctgcaggtcg actctagagg
     2941 atccccgggt accgagctcg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     3001 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     3061 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     3121 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt
     3181 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     3241 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3301 tgagaagaca agggacacga agtgatccgt ttccttgaca ggatatattg gcgggtaaac
     3361 taagtcgctg t
//
