LOCUS       sequence-490169-        4101 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  OsU6-2p::sgRNA scaffold in L1 vector backbone, for subcloning of 2nd
            20 bp target sequence. For use in monocots..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4101)
  AUTHORS   Abate BA, Hahn F, Chirivi D, Betti C, Fornara F, Molloy MC, Creasey 
            Krainer KM
  TITLE     Developing a Molecular Toolkit to ENABLE all to apply 
            CRISPR/Cas9-based Gene Editing in planta
  JOURNAL   bioRxiv 2025.11.09.687425
REFERENCE   2  (bases 1 to 4101)
  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 
            2025.11.09.687425"
COMMENT     Sequence Label: pGMF2-M
FEATURES             Location/Qualifiers
     source          1..4101
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     411..427
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    590..614
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     protein_bind    1032..1053
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1068..1073,1074..1091,1092..1098)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1106..1122
                     /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     1130..1146
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1142..1465
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1159..1215
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1216..1232)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1702..1726
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     primer_bind     complement(1880..1896)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1904..1920
                     /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)."
     promoter        complement(join(1928..1934,1935..1952,1953..1958))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1973..1994
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2111..2128)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2282..2870)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2362..2381)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3041..3832,3833..3901))
                     /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     3664..3683
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3902..4006)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4074..4092
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgcacga ggccctttca tctcgcgcgt ttcggtgatg acggtgaaaa
       61 cctctgacac atgcagctcc cctagacggt cacagcttgt ctgtaagcgg atgccgggag
      121 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
      181 tgcggcatca gagcagattg tactgagagt gcaccaaatg cggtgtgaaa taccgcacag
      241 atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg
      301 ggaagggcga tcggtgcggg cctcatcgct attacgccag ctggcgaaag ggggatgtgc
      361 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
      421 ggccagtgca acgcgatgac gatggatagc gattcatcga tgagctgacc cgatcgccgc
      481 cgccggaggg ttgcgtttga gacaggcgac agattctgac tgatgggctg cctgtatcga
      541 gtggtgattt tgtgccgagc tgccggtcgg ggagctgttg gctggctggt ggcaggatat
      601 attgtggtgt aaacaaattg acgcttagac aacttaataa cacattgcgg acgtttttaa
      661 tgtactgggg tggatgcagt gggccccact ctgtgaagac aattacgaat tcccatgggg
      721 agggatcatg aaccaacggc ctggctgtat ttggtggttg tgtagggaga tggggagaag
      781 aaaagcccga ttctcttcgc tgtgatgggc tggatgcatg cgggggagcg ggaggcccaa
      841 gtacgtgcac ggtgagcggc ccacagggcg agtgtgagcg cgagaggcgg gaggaacagt
      901 ttagtaccac attgcccagc taactcgaac gcgaccaact tataaacccg cgcgctgtcg
      961 cttgtgttgt gagaccgcag ctggcacgac aggtttgccg actggaaagc gggcagtgag
     1021 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     1081 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     1141 tatgaccatg attacgccaa gcttgcatgc ctgcaggtcg actctagagg atccccgggt
     1201 accgagctcg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     1261 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     1321 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat
     1381 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
     1441 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     1501 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgacggtctc
     1561 agtttcagag ctatgctgga aacagcatag caagttgaaa taaggctagt ccgttatcaa
     1621 cttgaaaaag tggcaccgag tcggtgcttt ttttttcgct gggattgtct tctgcacgaa
     1681 gtggtttaaa ctatcagtgt ttgacaggat atattggcgg gtaaacctaa gagaaaagag
     1741 cgtttattag aataatcgga tatttaaaag ggcgtgaaaa ggtttatccg ttcgtccatt
     1801 tgtatgtgca tgccaaccac agggttcccc aatcagttct ggaccagcga gctgtgctgc
     1861 gactcgtggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     1921 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     1981 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     2041 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     2101 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     2161 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2221 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2281 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     2341 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     2401 gctctcctgt tccgaccctg tcgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     2461 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     2521 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     2581 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     2641 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     2701 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     2761 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     2821 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     2881 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     2941 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3001 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3061 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3121 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3181 gagatccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3241 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3301 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     3361 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     3421 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     3481 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     3541 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     3601 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     3661 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     3721 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     3781 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     3841 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     3901 tactctacct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     3961 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4021 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     4081 gtatcacgag gccctttcat c
//
