LOCUS       sequence-383687-        8543 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MoClo Golden Gate Level 1 Position 5. Overexpression cassette of 
            Growth-Regulating Factor 4 (GRF4) plus GRF-Interacting Factor 1 
            (GRF4-GIF1). Improves in vitro wheat regeneration and 
            transformation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8543)
  TITLE     Golden Gate Level 1 plasmids containing the wheat GRF4-GIF1 gene 
            fusion
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8543)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pL1P5 ZmUbiP:GRF-GIF:NosT
FEATURES             Location/Qualifiers
     source          1..8543
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    42..66
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(222..1370)
                     /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(1706..2294)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1786..1805)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2465..3256,3257..3325))
                     /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     3088..3107
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3326..3430)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3517..4148
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4240..4264
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     promoter        4368..6359
                     /label=Ubi promoter
                     /note="maize polyubiquitin gene promoter"
     terminator      8285..8537
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
ORIGIN
        1 tgtgttgtct tctgcacgaa gtggtttaaa ctatcagtgt ttgacaggat atattggcgg
       61 gtaaacctaa gagaaaagag cgtttattag aataatcgga tatttaaaag ggcgtgaaaa
      121 ggtttatccg ttcgtccatt tgtatgtgca tgccaaccac agggttcccc agatcaggcg
      181 ctggctgctg aacccccagc cggaactgac cccacaaggc cctagcgttt gcaatgcacc
      241 aggtcatcat tgacccaggc gtgttccacc aggccgctgc ctcgcaactc ttcgcaggct
      301 tcgccgacct gctcgcgcca cttcttcacg cgggtggaat ccgatccgca catgaggcgg
      361 aaggtttcca gcttgagcgg gtacggctcc cggtgcgagc tgaaatagtc gaacatccgt
      421 cgggccgtcg gcgacagctt gcggtacttc tcccatatga atttcgtgta gtggtcgcca
      481 gcaaacagca cgacgatttc ctcgtcgatc aggacctggc aacgggacgt tttcttgcca
      541 cggtccagga cgcggaagcg gtgcagcagc gacaccgatt ccaggtgccc aacgcggtcg
      601 gacgtgaagc ccatcgccgt cgcctgtagg cgcgacaggc attcctcggc cttcgtgtaa
      661 taccggccat tgatcgacca gcccaggtcc tggcaaagct cgtagaacgt gaaggtgatc
      721 ggctcgccga taggggtgcg cttcgcgtac tccaacacct gctgccacac cagttcgtca
      781 tcgtcggccc gcagctcgac gccggtgtag gtgatcttca cgtccttgtt gacgtggaaa
      841 atgaccttgt tttgcagcgc ctcgcgcggg attttcttgt tgcgcgtggt gaacagggca
      901 gagcgggccg tgtcgtttgg catcgctcgc atcgtgtccg gccacggcgc aatatcgaac
      961 aaggaaagct gcatttcctt gatctgctgc ttcgtgtgtt tcagcaacgc ggcctgcttg
     1021 gcctcgctga cctgttttgc caggtcctcg ccggcggttt ttcgcttctt ggtcgtcata
     1081 gttcctcgcg tgtcgatggt catcgacttc gccaaacctg ccgcctcctg ttcaagacga
     1141 cgcgaacgct ccacggcggc cgatggcgcg ggcagggcag ggggagccag ttgcacgctg
     1201 tcgcgctcga tcttggccgt agcttgctgg accatcgagc cgacggactg gaaggtttcg
     1261 cggggcgcac gcatgacggt gcggcttgcg atggtttcgg catcctcggc ggaaaacccc
     1321 gcgtcgatca gttcttgcct gtatgccttc cggtcaaacg tccgattcat tcaccctcct
     1381 tgcgggattg ccccgactca cgccggggca atgtgccctt attcctgatt tgacccgcct
     1441 ggtgccttgg tgtccagata atccacctta tcggcaatga agtcggtccc gtagaccgtc
     1501 tggccgtcct tctcgtactt ggtattccga atcttgccct gcacgaatac cagcgacccc
     1561 ttgcccaaat acttgccgtg ggcctcggcc tgagagccaa aacacttgat gcggaagaag
     1621 tcggtgcgct cctgcttgtc gccggcatcg ttgcgccaca tctaggatct gccaggaacc
     1681 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     1741 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     1801 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     1861 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     1921 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     1981 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2041 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     2101 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     2161 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     2221 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     2281 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     2341 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     2401 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     2461 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     2521 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     2581 gccccagtgc tgcaatgata ccgcgagaac cacgctcacc ggctccagat ttatcagcaa
     2641 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     2701 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     2761 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     2821 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     2881 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     2941 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3001 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3061 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     3121 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     3181 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     3241 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     3301 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     3361 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     3421 gggttccgcg cacgaattgg ccagcgctgc catttttggg gtgaggccgt tcgcggccga
     3481 ggggcgcagc ccctgggggg atgggaggcc cgcgttagcg ggccgggagg gttcgagaag
     3541 ggggggcacc ccccttcggc gtgcgcggtc acgcgcacag ggcgcagccc tggttaaaaa
     3601 caaggtttat aaatattggt ttaaaagcag gttaaaagac aggttagcgg tggccgaaaa
     3661 acgggcggaa acccttgcaa atgctggatt ttctgcctgt ggacagcccc tcaaatgtca
     3721 ataggtgcgc ccctcatctg tcagcactct gcccctcaag tgtcaaggat cgcgcccctc
     3781 atctgtcagt agtcgcgccc ctcaagtgtc aataccgcag ggcacttatc cccaggcttg
     3841 tccacatcat ctgtgggaaa ctcgcgtaaa atcaggcgtt ttcgccgatt tgcgaggctg
     3901 gccagctcca cgtcgccggc cgaaatcgag cctgcccctc atctgtcaac gccgcgccgg
     3961 gtgagtcggc ccctcaagtg tcaacgtccg cccctcatct gtcagtgagg gccaagtttt
     4021 ccgcgaggta tccacaacgc cggcggccgc ggtgtctcgc acacggcttc gacggcgttt
     4081 ctggcgcgtt tgcagggcca tagacggccg ccagcccagc ggcgagggca accagcccgg
     4141 tgagcgtcgc aaaggagatc ctgatctgac tgatgggctg cctgtatcga gtggtgattt
     4201 tgtgccgagc tgccggtcgg ggagctgttg gctggctggt ggcaggatat attgtggtgt
     4261 aaacaaattg acgcttagac aacttaataa cacattgcgg acgtttttaa tgtactgggg
     4321 tggatgcagt gggccccact ctgtgaagac aacagagaat tcgcatgcgg aggtgcagcg
     4381 tgacccggtc gtgcccctct ctagagataa tgagcattgc atgtctaagt tataaaaaat
     4441 taccacatat tttttttgtc acacttgttt gaagtgcagt ttatctatct ttatacatat
     4501 atttaaactt tactctacga ataatataat ctatagtact acaataatat cagtgtttta
     4561 gagaatcata taaatgaaca gttagacatg gtctaaagga caattgagta ttttgacaac
     4621 aggactctac agttttatct ttttagtgtg catgtgttct cctttttttt tgcaaatagc
     4681 ttcacctata taatacttca tccattttat tagtacatcc atttagggtt tagggttaat
     4741 ggtttttata gactaatttt tttagtacat ctattttatt ctattttagc ctctaaatta
     4801 agaaaactaa aactctattt tagttttttt atttaataat ttagatataa aatagaataa
     4861 aataaagtga ctaaaaatta aacaaatacc ctttaagaaa ttaaaaaaac taaggaaaca
     4921 tttttcttgt ttcgagtaga taatgccagc ctgttaaacg ccgtcgacga gtctaacgga
     4981 caccaaccag cgaaccagca gcgtcgcgtc gggccaagcg aagcagacgg cacggcatct
     5041 ctgtcgctgc ctctggaccc ctctcgagag ttccgctcca ccgttggact tgctccgctg
     5101 tcggcatcca gaaattgcgt ggcggagcgg cagacgtgag ccggcacggc aggcggcctc
     5161 ctcctcctct cacggcacgg cagctacggg ggattccttt cccaccgctc cttcgctttc
     5221 ccttcctcgc ccgccgtaat aaatagacac cccctccaca ccctctttcc ccaacctcgt
     5281 gttgttcgga gcgcacacac acacaaccag atctccccca aatccacccg tcggcacctc
     5341 cgcttcaagg tacgccgctc gtcctccccc cccccccctc tctaccttct ctagatcggc
     5401 gttccggtcc atggttaggg cccggtagtt ctacttctgt tcatgtttgt gttagatccg
     5461 tgtttgtgtt agatccgtgc tgctagcgtt cgtacacgga tgcgacctgt acgtcagaca
     5521 cgttctgatt gctaacttgc cagtgtttct ctttggggaa tcctgggatg gctctagccg
     5581 ttccgcagac gggatcgatt tcatgatttt ttttgtttcg ttgcataggg tttggtttgc
     5641 ccttttcctt tatttcaata tatgccgtgc acttgtttgt cgggtcatct tttcatgctt
     5701 ttttttgtct tggttgtgat gatgtggtct ggttgggcgg tcgttctaga tcggagtaga
     5761 attctgtttc aaactacctg gtggatttat taattttgga tctgtatgtg tgtgccatac
     5821 atattcatag ttacgaattg aagatgatgg atggaaatat cgatctagga taggtataca
     5881 tgttgatgcg ggttttactg atgcatatac agagatgctt tttgttcgct tggttgtgat
     5941 gatgtggtgt ggttgggcgg tcgttcattc gttctagatc ggagtagaat actgtttcaa
     6001 actacctggt gtatttatta attttggaac tgtatgtgtg tgtcatacat cttcatagtt
     6061 acgagtttaa gatggatgga aatatcgatc taggataggt atacatgttg atgtgggttt
     6121 tactgatgca tatacatgat ggcatatgca gcatctattc atatgctcta accttgagta
     6181 cctatctatt ataataaaca agtatgtttt ataattattt tgatcttgat atacttggat
     6241 gatggcatat gcagcagcta tatgtggatt tttttagccc tgccttcata cgctatttat
     6301 ttgcttggta ctgtttcttt tgtcgatgct caccctgttg tttggtgtta cttctgcagg
     6361 aatggcgatg ccgtatgcct ctctttcccc ggcaggcgac cgccgctcct ccccggccgc
     6421 caccgccacc gcctccctcc tccccttctg ccgctcctcc cccttctccg ccggcggcaa
     6481 tggcggcatg ggggaggagg cgcggatgga cgggaggtgg atggcgaggc cggtgccctt
     6541 cacggcggcg cagtacgagg agctggagca ccaggcgctc atatacaagt acctggtggc
     6601 cggcgtgtcc gtcccgccgg atctcgtgct ccccatccgc cgcggcatcg agtccctcgc
     6661 cgcccgcttc taccacaacc ccctcgccat cgggtacgga tcgtacctgg gcaagaaggt
     6721 ggatccggag ccgggccggt gccggcgcac ggacggcaag aagtggcggt gcgccaagga
     6781 ggccgcctcc gactccaagt attgcgagcg ccacatgcac cgcggccgca accgttcaag
     6841 aaagcctgtg gaaacgcagc tcgtctcgca ctcccagccg ccggccgcct ccgtcgtgcc
     6901 gcccctcgcc accggcttcc acaaccactc cctctacccc gccatcggcg gcaccaacgg
     6961 tggtggaggc ggggggaaca acggcatgtc catgcccggc acgttctcct ccgcgctggg
     7021 gccgcctcag cagcacatgg gcaacaatgc cgcctctccc tacgcggctc tcggcggcgc
     7081 cggaacatgc aaagatttca ggtataccgc atatggaata agatctttgg cagacgagca
     7141 gagtcagctc atgacagaag ccatgaacac ctccgtggag aacccatggc gcctgccgcc
     7201 atcttctcaa acgactacat tcccgctctc aagctactct cctcagcttg gagcaacgag
     7261 tgacctgggt cagaacaaca gcagcaacaa caacagcggc gtcaaggccg agggacagca
     7321 gcagcagcag ccgctctcct tcccggggtg cggcgacttc ggcagcggcg actccgcgaa
     7381 gcaggagaac cagacgctgc ggccgttctt cgacgagtgg cccaagacga gggactcgtg
     7441 gtcggacctg accgacgaca actcgaacgt cgcctccttc tcggccaccc agctgtcgat
     7501 ctcgataccc atgacgtcct ccgacttctc cgccgccagc tcccagtcgc ccaacggcat
     7561 gctgttcgcc ggcgagatgt acgcggccgc tgccatgcag cagcaacacc tgatgcagat
     7621 gaaccagagc atgatggggg gctacgcttc ctctaccact gccaccactg atctcattca
     7681 gcagtacctg gatgagaaca agcagctgat cctggccatc ctcgacaacc agaacaacgg
     7741 caaggtggag gagtgcgcac ggaaccaagc taagctccag cagaacctca tgtacctcgc
     7801 cgccatcgcc gacagccagc ctccgcagac ggcatcgctg tctcagtacc cgtccaacct
     7861 gatgatgcag tccgggccgc ggtacatgca gcagcagtcg gcgcagatga tgtcgccgca
     7921 gtcgctgatg gcggcgcggt cgtcgatgat gtacgcgcag caggccatgt cgccgctcca
     7981 gcagcagcag cagcagcagc agcaccaggc ggccgcgcac ggccagctgg ggatgtcctc
     8041 cggcgcgacc accgggttca acctcctgca cggtgaggcc agcatgggcg gcggcggcgg
     8101 cgccactggc aacagcatga tgaacgccag cgtcttttcg gactatggcc gcggcggcag
     8161 cggcgccaag gaggggtcga cctcgctgtc ggccgacgct cgcggcgcca actctggcgc
     8221 gcacagcggc gacggggagt acctcaaggg caccgaggag gaaggaagct aggcttgtca
     8281 agcagatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg ttgccggtct
     8341 tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta
     8401 atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat tatacattta
     8461 atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc
     8521 atctatgtta ctagatcgac gct
//
