LOCUS       sequence-225200-        4685 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PCR template for sgRNA classic with polyT as terminator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4685)
  AUTHORS   Castel B, Tomlinson L, Locci F, Yang Y, Jones JDG
  TITLE     Optimization of T-DNA architecture for Cas9-mediated mutagenesis in 
            Arabidopsis.
  JOURNAL   PLoS One. 2019 Jan 9;14(1):e0204778. doi: 
            10.1371/journal.pone.0204778. eCollection 2019.
  PUBMED    30625150
REFERENCE   2  (bases 1 to 4685)
  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: "PLoS One.";
            date: "2019-01-9"; volume: "14(1):e0204778. doi"; pages: " 
            10.1371/journal.pone.0204778. eCollection 2019"
COMMENT     Sequence Label: LBJJ383
FEATURES             Location/Qualifiers
     source          1..4685
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        260..335
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    388..412
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(568..1716)
                     /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(2052..2640)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2132..2151)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2811..3602,3603..3671))
                     /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     3434..3453
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3672..3776)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3863..4494
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4586..4610
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 tctgtgaaga caaactagaa ttcgagctcg gagcatcttc attcttaaga tatgaagata
       61 atcttcaaaa ggcccctggg aatctgaaag aagagaagca ggcccattta tatgggaaag
      121 aacaatagta tttcttatat aggcccattt aagttgaaaa caatcttcaa aagtcccaca
      181 tcgcttagat aagaaaacga agctgagttt atatacagct agagtcgaag tagtgattgc
      241 gtatcttcgg ccatgaagcg ttttagagct agaaatagca agttaaaata aggctagtcc
      301 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttcgctttac ttgtcttctg
      361 cacgaagtgg tttaaactat cagtgtttga caggatatat tggcgggtaa acctaagaga
      421 aaagagcgtt tattagaata atcggatatt taaaagggcg tgaaaaggtt tatccgttcg
      481 tccatttgta tgtgcatgcc aaccacaggg ttccccagat caggcgctgg ctgctgaacc
      541 cccagccgga actgacccca caaggcccta gcgtttgcaa tgcaccaggt catcattgac
      601 ccaggcgtgt tccaccaggc cgctgcctcg caactcttcg caggcttcgc cgacctgctc
      661 gcgccacttc ttcacgcggg tggaatccga tccgcacatg aggcggaagg tttccagctt
      721 gagcgggtac ggctcccggt gcgagctgaa atagtcgaac atccgtcggg ccgtcggcga
      781 cagcttgcgg tacttctccc atatgaattt cgtgtagtgg tcgccagcaa acagcacgac
      841 gatttcctcg tcgatcagga cctggcaacg ggacgttttc ttgccacggt ccaggacgcg
      901 gaagcggtgc agcagcgaca ccgattccag gtgcccaacg cggtcggacg tgaagcccat
      961 cgccgtcgcc tgtaggcgcg acaggcattc ctcggccttc gtgtaatacc ggccattgat
     1021 cgaccagccc aggtcctggc aaagctcgta gaacgtgaag gtgatcggct cgccgatagg
     1081 ggtgcgcttc gcgtactcca acacctgctg ccacaccagt tcgtcatcgt cggcccgcag
     1141 ctcgacgccg gtgtaggtga tcttcacgtc cttgttgacg tggaaaatga ccttgttttg
     1201 cagcgcctcg cgcgggattt tcttgttgcg cgtggtgaac agggcagagc gggccgtgtc
     1261 gtttggcatc gctcgcatcg tgtccggcca cggcgcaata tcgaacaagg aaagctgcat
     1321 ttccttgatc tgctgcttcg tgtgtttcag caacgcggcc tgcttggcct cgctgacctg
     1381 ttttgccagg tcctcgccgg cggtttttcg cttcttggtc gtcatagttc ctcgcgtgtc
     1441 gatggtcatc gacttcgcca aacctgccgc ctcctgttca agacgacgcg aacgctccac
     1501 ggcggccgat ggcgcgggca gggcaggggg agccagttgc acgctgtcgc gctcgatctt
     1561 ggccgtagct tgctggacca tcgagccgac ggactggaag gtttcgcggg gcgcacgcat
     1621 gacggtgcgg cttgcgatgg tttcggcatc ctcggcggaa aaccccgcgt cgatcagttc
     1681 ttgcctgtat gccttccggt caaacgtccg attcattcac cctccttgcg ggattgcccc
     1741 gactcacgcc ggggcaatgt gcccttattc ctgatttgac ccgcctggtg ccttggtgtc
     1801 cagataatcc accttatcgg caatgaagtc ggtcccgtag accgtctggc cgtccttctc
     1861 gtacttggta ttccgaatct tgccctgcac gaataccagc gaccccttgc ccaaatactt
     1921 gccgtgggcc tcggcctgag agccaaaaca cttgatgcgg aagaagtcgg tgcgctcctg
     1981 cttgtcgccg gcatcgttgc gccacatcta ggatctgcca ggaaccgtaa aaaggccgcg
     2041 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     2101 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     2161 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     2221 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     2281 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     2341 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     2401 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     2461 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     2521 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     2581 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     2641 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     2701 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     2761 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     2821 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     2881 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     2941 atgataccgc gagaaccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     3001 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     3061 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     3121 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     3181 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     3241 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     3301 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     3361 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     3421 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     3481 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     3541 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     3601 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     3661 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaagg ttattgtctc
     3721 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcacg
     3781 aattggccag cgctgccatt tttggggtga ggccgttcgc ggccgagggg cgcagcccct
     3841 ggggggatgg gaggcccgcg ttagcgggcc gggagggttc gagaaggggg ggcacccccc
     3901 ttcggcgtgc gcggtcacgc gcacagggcg cagccctggt taaaaacaag gtttataaat
     3961 attggtttaa aagcaggtta aaagacaggt tagcggtggc cgaaaaacgg gcggaaaccc
     4021 ttgcaaatgc tggattttct gcctgtggac agcccctcaa atgtcaatag gtgcgcccct
     4081 catctgtcag cactctgccc ctcaagtgtc aaggatcgcg cccctcatct gtcagtagtc
     4141 gcgcccctca agtgtcaata ccgcagggca cttatcccca ggcttgtcca catcatctgt
     4201 gggaaactcg cgtaaaatca ggcgttttcg ccgatttgcg aggctggcca gctccacgtc
     4261 gccggccgaa atcgagcctg cccctcatct gtcaacgccg cgccgggtga gtcggcccct
     4321 caagtgtcaa cgtccgcccc tcatctgtca gtgagggcca agttttccgc gaggtatcca
     4381 caacgccggc ggccgcggtg tctcgcacac ggcttcgacg gcgtttctgg cgcgtttgca
     4441 gggccataga cggccgccag cccagcggcg agggcaacca gcccggtgag cgtcgcaaag
     4501 gagatcctga tctgactgat gggctgcctg tatcgagtgg tgattttgtg ccgagctgcc
     4561 ggtcggggag ctgttggctg gctggtggca ggatatattg tggtgtaaac aaattgacgc
     4621 ttagacaact taataacaca ttgcggacgt ttttaatgta ctggggtgga tgcagtgggc
     4681 cccac
//
