LOCUS       sequence-146014-        3222 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tRNA and scaffold for the assembly of GBoligomers for the first 
            position (positon [M1_n-1]) of a polycistronic tRNA-gRNA regulated 
            by the (monocot) U3 promoter (2-part multiplexing).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3222)
  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 3222)
  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 [M1_n-1] (GB1210)
FEATURES             Location/Qualifiers
     source          1..3222
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(174..278)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(356..811)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(493..514)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     705..724
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     937..959
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     951..968
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     952..968
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..994
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        975..993
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1024..1033
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1028..1037
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        1168..1243
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1338..1356)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1339..1356)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1374..1390)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1374..1390)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1387..1409)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1398..1414
                     /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(1422..1428,1429..1446,1447..1452))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1467..1488
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1605..1622)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1776..2364)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1856..1875)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2535..3222,1..104,105..173))
                     /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     3158..3177
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
       61 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
      121 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
      181 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
      241 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
      301 cctgatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt
      361 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
      421 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
      481 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
      541 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
      601 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
      661 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
      721 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
      781 gccgcgctta atgcgccgct acagggcgcg tccattcgcc attcaggctg cgcaactgtt
      841 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
      901 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
      961 cggccagtga attgtaatac gactcactat agggcgaatt gggcccgacg tcgcatgctc
     1021 ccggccgcca tggcggccgc gggaattcga ttcccgtctc gctcgggcaa acaaagcacc
     1081 agtggtctag tggtagaata gtaccctgcc acggtacaga cccgggttcg attcccggct
     1141 ggtgcagaga cggcgcgcgc cgtctcagtt ttagagctag aaatagcaag ttaaaataag
     1201 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcaatgagc gagacgggat
     1261 cactagtgaa ttcgcggccg cctgcaggtc gaccatatgg gagagctccc aacgcgttgg
     1321 atgcatagct tgagtattct atagtgtcac ctaaatagct tggcgtaatc atggtcatag
     1381 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     1441 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     1501 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     1561 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     1621 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     1681 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     1741 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     1801 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     1861 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     1921 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     1981 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     2041 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     2101 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     2161 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     2221 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     2281 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     2341 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     2401 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     2461 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     2521 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     2581 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     2641 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     2701 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     2761 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     2821 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     2881 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     2941 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     3001 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     3061 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     3121 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     3181 actttaaaag tgctcatcat tggaaaacgt tcttcggggc ga
//
