LOCUS       sequence-313824-        3188 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR/Cas9 genome editing in plants; a template for PCR-derived 
            fragments used in the assembly of polycistronic transcripts, where 
            sgRNAs are separated by an Arabidopsis alanine tRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3188)
  AUTHORS   Angulo J, Astin CP, Bauer O , Blash KJ, Bowen NM, Chukwudinma NJ, 
            Dinofrio AS, Faletti DO, Ghulam AM, Gusinde-Duffy CM, Horace KJ, 
            Ingram AM, Isaack KE, Kiser RJ, Kobylanski JS, Long MR, Manning GA, 
            Morales JM, Nguyen KH, Pham RT, Phillips MH, Reel TW, Seo JE, Vo HD,
            Wukuson AM, Yeary KA, Zheng GY, Lukowitz W
  TITLE     Targeted mutagenesis of the Arabidopsis GROWTH-REGULATING FACTOR 
            (GRF) gene family suggests competition of multiplexed sgRNAs for 
            Cas9 apoprotein
  JOURNAL   bioRxiv
REFERENCE   2  (bases 1 to 3188)
  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: "bioRxiv"
COMMENT     Sequence Label: pGEM-2t
FEATURES             Location/Qualifiers
     source          1..3188
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1..18
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_RNA        100..175
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(329..348)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(330..348)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(355..372)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(355..371)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(364..386)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      512..967
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(599..618)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     809..830
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1045..1149
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1150..1218,1219..2010)
                     /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     complement(1368..1387)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2181..2769
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2670..2689
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2923..2940
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3057..3078
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3093..3098,3099..3116,3117..3123)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3131..3147
                     /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     3136..3158
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3155..3171
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3155..3171
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 atttaggtga cactatagaa tactcaagct atgcatccaa cgcgttggga gctctccgga
       61 tccaagcttg gtctcgattg tttctcacgc gagcaattcg ttttagagct agaaatagca
      121 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcaacaa
      181 agcaccagtg gtctagtggt agaatagtac cctgccacgg tacagacccg ggttcgattc
      241 ccggctggtg cagatgtgac caaccagttc ctgtttagag accgaattcc tcgagtctag
      301 aggagcatgc gacgtcgggc ccaattcgcc ctatagtgag tcgtattaca attcactggc
      361 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
      421 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
      481 ccaacagttg cgcagcctga atggcgaatg gacgcgccct gtagcggcgc attaagcgcg
      541 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
      601 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
      661 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
      721 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
      781 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
      841 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
      901 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
      961 acaatttcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatca
     1021 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     1081 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     1141 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     1201 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     1261 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     1321 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     1381 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     1441 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     1501 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     1561 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     1621 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     1681 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     1741 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     1801 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     1861 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     1921 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     1981 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     2041 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     2101 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2161 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2221 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2281 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     2341 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2401 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     2461 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2521 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     2581 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     2641 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     2701 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     2761 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     2821 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     2881 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     2941 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     3001 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     3061 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     3121 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     3181 gccaagct
//
