LOCUS       sequence-490165-        3526 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AtU6-26p::sgRNA scaffold in L1 vector backbone, for subcloning of 
            2nd 20 bp target sequence. For use in dicots..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3526)
  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 3526)
  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-D
FEATURES             Location/Qualifiers
     source          1..3526
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    76..100
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     protein_bind    720..741
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(756..761,762..779,780..786)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    794..810
                     /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     799..821
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     818..834
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     818..834
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             830..1153
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    847..903
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(904..921)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(904..920)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(913..935)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1129..1148)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    1390..1414
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     primer_bind     complement(1536..1553)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1707..2295)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1787..1806)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2466..3257,3258..3326))
                     /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     3089..3108
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3327..3431)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3499..3517
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tctgactgat gggctgcctg tatcgagtgg tgattttgtg ccgagctgcc ggtcggggag
       61 ctgttggctg gctggtggca ggatatattg tggtgtaaac aaattgacgc ttagacaact
      121 taataacaca ttgcggacgt ttttaatgta ctggggtgga tgcagtgggc cccactctgt
      181 gaagacaatt acgaattccc atggggagaa gcttcgttga acaacggaaa ctcgacttgc
      241 cttccgcaca atacatcatt tcttcttagc tttttttctt cttcttcgtt catacagttt
      301 ttttttgttt atcagcttac attttcttga accgtagctt tcgttttctt ctttttaact
      361 ttccattcgg agtttttgta tcttgtttca tagtttgtcc caggattaga atgattaggc
      421 atcgaacctt caagaatttg attgaataaa acatcttcat tcttaagata tgaagataat
      481 cttcaaaagg cccctgggaa tctgaaagaa gagaagcagg cccatttata tgggaaagaa
      541 caatagtatt tcttatatag gcccatttaa gttgaaaaca atcttcaaaa gtcccacatc
      601 gcttagataa gaaaacgaag ctgagtttat atacagctag agtcgaagta gtgattgtga
      661 gaccgcagct ggcacgacag gtttgccgac tggaaagcgg gcagtgagcg caacgcaatt
      721 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
      781 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
      841 tacgccaagc ttgcatgcct gcaggtcgac tctagaggat ccccgggtac cgagctcgaa
      901 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
      961 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     1021 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct
     1081 ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc
     1141 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     1201 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg acggtctcag tttcagagct
     1261 atgctggaaa cagcatagca agttgaaata aggctagtcc gttatcaact tgaaaaagtg
     1321 gcaccgagtc ggtgcttttt ttttcgctgg gattgtcttc tgcacgaagt ggtttaaact
     1381 atcagtgttt gacaggatat attggcgggt aaacctaaga gaaaagagcg tttattagaa
     1441 taatcggata tttaaaaggg cgtgaaaagg tttatccgtt cgtccatttg tatgtgcatg
     1501 ccaaccacag ggttccccag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     1561 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     1621 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     1681 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     1741 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     1801 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     1861 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     1921 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     1981 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     2041 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     2101 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     2161 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     2221 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     2281 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     2341 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     2401 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     2461 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     2521 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     2581 ggccccagtg ctgcaatgat accgcgagat ccacgctcac cggctccaga tttatcagca
     2641 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     2701 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     2761 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     2821 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     2881 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     2941 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     3001 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     3061 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     3121 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     3181 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     3241 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     3301 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     3361 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     3421 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     3481 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
