LOCUS       sequence-331267-        7273 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GoldenGate (MoClo) Level 1 Position 1 (Reverse) hygromycin selection
            cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7273)
  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 7273)
  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: Level 1 P1 OsActinP:Hygint:NosT
FEATURES             Location/Qualifiers
     source          1..7273
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    887..911
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(1067..2215)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      complement(2551..3139)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2631..2650)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3310..4101,4102..4170))
                     /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     3933..3952
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4171..4275)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4362..4993
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    5085..5109
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     terminator      complement(5220..5472)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     intron          6172..6361
                     /label=cat1 intron
                     /note="castor bean catalase intron, modified"
     CDS             complement(7243..7260)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(join(7263..7273,1..832))
                     /label=Act1 promoter
                     /note="rice actin promoter"
ORIGIN
        1 gatgggagga gagggggggg tatgtatata gtggcgatgg ggggcgtttc tttggaagcg
       61 gagggagggc cggcctcgtc gctggctcgc gatcctcctc gcgtttccgg cccccacgac
      121 ccggacccac ctgctgtttt ttctttttct tttttttctt tctttttttt tttttggctg
      181 cgagacgtgc ggtgcgtgcg gacaactcac ggtgatagtg ggggggtgtg gagactattg
      241 tccagttggc tggactgggg tgggttgggt tgggttgggt tgggctgggc ttgctatgga
      301 tcgtggatag cactttgggc tttaggaact ttaggggttg tttttgtaaa tgttttgagt
      361 ctaagtttat cttttatttt tactagaaaa aatacccatg cgctgcaacg ggggaaagct
      421 attttaatct tattattgtt cattgtgaga attcgcctga atatatattt ttctcaaaaa
      481 ttatgtcaaa ttagcatatg ggttttttta aagatatttc ttatacaaat ccctctgtat
      541 ttacaaaagc aaacgaactt aaaacccgac tcaaatacag atatgcattt ccaaaagcga
      601 ataaacttaa aaaccaattc atacaaaaat gacgtatcaa agtaccgaca aaaacatcct
      661 caatttttat aatagtagaa aagagtaaat ttcactttgg gccacctttt attaccgata
      721 ttttacttta taccaccttt taactgatgt tttcactttt gaccaggtaa tcttaccttt
      781 gttttatttt ggactatccc gactctcttc tcaagcatat gaatgacctc gagtatgcta
      841 gctccgcaat tgtcttctgc acgaagtggt ttaaactatc agtgtttgac aggatatatt
      901 ggcgggtaaa cctaagagaa aagagcgttt attagaataa tcggatattt aaaagggcgt
      961 gaaaaggttt atccgttcgt ccatttgtat gtgcatgcca accacagggt tccccagatc
     1021 aggcgctggc tgctgaaccc ccagccggaa ctgaccccac aaggccctag cgtttgcaat
     1081 gcaccaggtc atcattgacc caggcgtgtt ccaccaggcc gctgcctcgc aactcttcgc
     1141 aggcttcgcc gacctgctcg cgccacttct tcacgcgggt ggaatccgat ccgcacatga
     1201 ggcggaaggt ttccagcttg agcgggtacg gctcccggtg cgagctgaaa tagtcgaaca
     1261 tccgtcgggc cgtcggcgac agcttgcggt acttctccca tatgaatttc gtgtagtggt
     1321 cgccagcaaa cagcacgacg atttcctcgt cgatcaggac ctggcaacgg gacgttttct
     1381 tgccacggtc caggacgcgg aagcggtgca gcagcgacac cgattccagg tgcccaacgc
     1441 ggtcggacgt gaagcccatc gccgtcgcct gtaggcgcga caggcattcc tcggccttcg
     1501 tgtaataccg gccattgatc gaccagccca ggtcctggca aagctcgtag aacgtgaagg
     1561 tgatcggctc gccgataggg gtgcgcttcg cgtactccaa cacctgctgc cacaccagtt
     1621 cgtcatcgtc ggcccgcagc tcgacgccgg tgtaggtgat cttcacgtcc ttgttgacgt
     1681 ggaaaatgac cttgttttgc agcgcctcgc gcgggatttt cttgttgcgc gtggtgaaca
     1741 gggcagagcg ggccgtgtcg tttggcatcg ctcgcatcgt gtccggccac ggcgcaatat
     1801 cgaacaagga aagctgcatt tccttgatct gctgcttcgt gtgtttcagc aacgcggcct
     1861 gcttggcctc gctgacctgt tttgccaggt cctcgccggc ggtttttcgc ttcttggtcg
     1921 tcatagttcc tcgcgtgtcg atggtcatcg acttcgccaa acctgccgcc tcctgttcaa
     1981 gacgacgcga acgctccacg gcggccgatg gcgcgggcag ggcaggggga gccagttgca
     2041 cgctgtcgcg ctcgatcttg gccgtagctt gctggaccat cgagccgacg gactggaagg
     2101 tttcgcgggg cgcacgcatg acggtgcggc ttgcgatggt ttcggcatcc tcggcggaaa
     2161 accccgcgtc gatcagttct tgcctgtatg ccttccggtc aaacgtccga ttcattcacc
     2221 ctccttgcgg gattgccccg actcacgccg gggcaatgtg cccttattcc tgatttgacc
     2281 cgcctggtgc cttggtgtcc agataatcca ccttatcggc aatgaagtcg gtcccgtaga
     2341 ccgtctggcc gtccttctcg tacttggtat tccgaatctt gccctgcacg aataccagcg
     2401 accccttgcc caaatacttg ccgtgggcct cggcctgaga gccaaaacac ttgatgcgga
     2461 agaagtcggt gcgctcctgc ttgtcgccgg catcgttgcg ccacatctag gatctgccag
     2521 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     2581 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     2641 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     2701 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     2761 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     2821 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     2881 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     2941 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     3001 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3061 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     3121 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     3181 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     3241 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     3301 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     3361 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     3421 atctggcccc agtgctgcaa tgataccgcg agaaccacgc tcaccggctc cagatttatc
     3481 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     3541 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     3601 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     3661 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     3721 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     3781 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     3841 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     3901 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     3961 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     4021 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     4081 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     4141 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     4201 ttatcaaggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     4261 aataggggtt ccgcgcacga attggccagc gctgccattt ttggggtgag gccgttcgcg
     4321 gccgaggggc gcagcccctg gggggatggg aggcccgcgt tagcgggccg ggagggttcg
     4381 agaagggggg gcacccccct tcggcgtgcg cggtcacgcg cacagggcgc agccctggtt
     4441 aaaaacaagg tttataaata ttggtttaaa agcaggttaa aagacaggtt agcggtggcc
     4501 gaaaaacggg cggaaaccct tgcaaatgct ggattttctg cctgtggaca gcccctcaaa
     4561 tgtcaatagg tgcgcccctc atctgtcagc actctgcccc tcaagtgtca aggatcgcgc
     4621 ccctcatctg tcagtagtcg cgcccctcaa gtgtcaatac cgcagggcac ttatccccag
     4681 gcttgtccac atcatctgtg ggaaactcgc gtaaaatcag gcgttttcgc cgatttgcga
     4741 ggctggccag ctccacgtcg ccggccgaaa tcgagcctgc ccctcatctg tcaacgccgc
     4801 gccgggtgag tcggcccctc aagtgtcaac gtccgcccct catctgtcag tgagggccaa
     4861 gttttccgcg aggtatccac aacgccggcg gccgcggtgt ctcgcacacg gcttcgacgg
     4921 cgtttctggc gcgtttgcag ggccatagac ggccgccagc ccagcggcga gggcaaccag
     4981 cccggtgagc gtcgcaaagg agatcctgat ctgactgatg ggctgcctgt atcgagtggt
     5041 gattttgtgc cgagctgccg gtcggggagc tgttggctgg ctggtggcag gatatattgt
     5101 ggtgtaaaca aattgacgct tagacaactt aataacacat tgcggacgtt tttaatgtac
     5161 tggggtggat gcagtgggcc ccactctgtg aagacaatgc cgaattcgga tccagcgtcg
     5221 atctagtaac atagatgaca ccgcgcgcga taatttatcc tagtttgcgc gctatatttt
     5281 gttttctatc gcgtattaaa tgtataattg cgggactcta atcataaaaa cccatctcat
     5341 aaataacgtc atgcattaca tgttaattat tacatgctta acgtaattca acagaaatta
     5401 tatgataatc atcgcaagac cggcaacagg attcaatctt aagaaacttt attgccaaat
     5461 gtttgaacga tctgcttgac aagcctattc ctttgccctc ggacgagtgc tggggcgtcg
     5521 gtttccacta tcggcgagta cttctacaca gccatcggtc cagacggccg cgcttctgcg
     5581 ggcgatttgt gtacgcccga cagtcccggc tccggatcgg acgattgcgt cgcatcgacc
     5641 ctgcgcccaa gctgcatcat cgaaattgcc gtcaaccaag ctctgataga gttggtcaag
     5701 accaatgcgg agcatatacg cccggagccg cggcgatcct gcaagctccg gatgcctccg
     5761 ctcgaagtag cgcgtctgct gctccataca agccaaccac ggcctccaga agaagatgtt
     5821 ggcgacctcg tattgggaat ccccgaacat cgcctcgctc cagtcaatga ccgctgttat
     5881 gcggccattg tccgtcagga cattgttgga gccgaaatcc gcgtgcacga ggtgccggac
     5941 ttcggggcag tcctcggccc aaagcatcag ctcatcgaga gcctgcgcga cggacgcact
     6001 gacggtgtcg tccatcacag tttgccagtg atacacatgg ggatcagcaa tcgcgcatat
     6061 gaaatcacgc catgtagtgt attgaccgat tccttgcggt ccgaatgggc cgaacccgct
     6121 cgtctggcta agatcggccg cagcgatcgc atccatggcc tccgcgaccg gctgcagtta
     6181 tcatcatcat catagacaca cgaaataaag taatcagatt atcagttaaa gctatgtaat
     6241 atttacacca taaccaatca attaaaaaat agatcagttt aaagaaagat caaagctcaa
     6301 aaaaataaaa agagaaaagg gtcctaacca agaaaatgaa ggagaaaaac tagaaattta
     6361 cctgcagaac agcgggcagt tcggtttcag gcaggtcttg caacgtgaca ccctgtgcac
     6421 ggcgggagat gcaataggtc aggctctcgc tgaattcccc aatgtcaagc acttccggaa
     6481 tcgggagcgc ggccgatgca aagtgccgat aaacataacg atctttgtag aaaccatcgg
     6541 cgcagctatt tacccgcagg acatatccac gccctcctac atcgaagctg aaagcacgag
     6601 attcttcgcc ctccgagagc tgcatcaggt cggagacgct gtcgaacttt tcgatcagaa
     6661 acttctcgac agacgtcgcg gtgagttcag gctttttcat tggcttctac ctacaaaaaa
     6721 gctccgcacg aggctgcatt tgtcacaaat catgaaaaga aaaactaccg atgaacaatg
     6781 ctgagggatt caaattctac ccacaaaaag aagaaagaaa gatctagcac atctaagcct
     6841 gacgaagcag cagaaatata taaaaatata aaccatagtg cccttttccc ctcttcctga
     6901 tcttgtttag catggcggaa attttaaacc ccccatcatc tcccccaaca acggcggatc
     6961 gcagatctac atccgagagc cccattcccc gcgagatccg ggccggatcc acgccggcga
     7021 gagccccagc cgcgagatcc cgcccctccc gcgcaccgat ctgggcgcgc acgaagccgc
     7081 ctctcgccca cccaaactac caaggccaaa gatcgtgtcc gagacggaaa aaaaaaacgg
     7141 agaaagaaag aggagagggg cggggtggtt accggcgcgg cggcggcgga gggggagggg
     7201 ggaggagctc gtcgtccggc agcgaggggg gaggaggtgg aggtggtggt ggtggtggtg
     7261 gtagggttgg ggg
//
