LOCUS       sequence-146004-        3220 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tRNA and scaffold for the assembly of GBoligomers for the first 
            position (positon [D1_2]) of a polycistronic tRNA-gRNA regulated by 
            the dicot U6-26 or U6-1 promoter (3-part multiplexing).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3220)
  AUTHORS   Vazquez-Vilar M, Bernabe-Orts JM, Fernandez-Del-Carmen A, Ziarsolo 
            P, Blanca J, Granell A, Orzaez D
  TITLE     A modular toolbox for gRNA-Cas9 genome engineering in plants based 
            on the GoldenBraid standard.
  JOURNAL   Plant Methods. 2016 Feb 1;12:10. doi: 10.1186/s13007-016-0101-2. 
            eCollection 2016.
  PUBMED    26839579
REFERENCE   2  (bases 1 to 3220)
  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: "Plant 
            Methods."; date: "2016-02-1"; volume: "12:10. doi"; pages: " 
            10.1186/s13007-016-0101-2. eCollection 2016"
COMMENT     Sequence Label: tRNA-gRNA position [D1_2] (GB1205)
FEATURES             Location/Qualifiers
     source          1..3220
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        complement(9..84)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(279..297)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(280..297)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(315..331)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(315..331)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(328..350)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    339..355
                     /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(363..369,370..387,388..393))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    408..429
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(546..563)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(717..1305)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(797..816)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1476..2267,2268..2336))
                     /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     2099..2118
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2337..2441)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2519..2974)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2656..2677)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2868..2887
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3100..3122
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3114..3131
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3115..3131
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3138..3157
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3138..3156
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      3187..3196
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      3191..3200
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 ctcgctcagc accgactcgg tgccactttt tcaagttgat aacggactag ccttatttta
       61 acttgctatt tctagctcta aaactgagac ggcgcgcgcc gtctctgcac cagccgggaa
      121 tcgaacccgg gtctgtaccg tggcagggta ctattctacc actagaccac tggtgctttg
      181 ttcaatcgag cgagacggga tcactagtga attcgcggcc gcctgcaggt cgaccatatg
      241 ggagagctcc caacgcgttg gatgcatagc ttgagtattc tatagtgtca cctaaatagc
      301 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
      361 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
      421 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
      481 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
      541 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
      601 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
      661 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
      721 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
      781 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
      841 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
      901 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
      961 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     1021 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     1081 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     1141 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     1201 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     1261 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     1321 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     1381 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     1441 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     1501 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     1561 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     1621 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     1681 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     1741 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     1801 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     1861 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     1921 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     1981 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     2041 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     2101 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     2161 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     2221 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     2281 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     2341 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     2401 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     2461 ccacctgatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggaa
     2521 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     2581 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     2641 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     2701 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     2761 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     2821 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     2881 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     2941 cccgccgcgc ttaatgcgcc gctacagggc gcgtccattc gccattcagg ctgcgcaact
     3001 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     3061 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     3121 cgacggccag tgaattgtaa tacgactcac tatagggcga attgggcccg acgtcgcatg
     3181 ctcccggccg ccatggcggc cgcgggaatt cgattcccgt
//
