LOCUS       sequence-319681-        3231 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tRNA and scaffold for the assembly of GBoligomers for position 
            [D1_n] of a monocistronic tRNA-gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3231)
  AUTHORS   Vazquez-Vilar M, Garcia-Carpintero V, Selma S, Bernabe-Orts JM, 
            Sanchez-Vicente J, Salazar-Sarasua B, Ressa A, de Paola C, Ajenjo M,
            Quintela JC, Fernandez-Del-Carmen A, Granell A, Orzaez D
  TITLE     The GB4.0 Platform, an All-In-One Tool for CRISPR/Cas-Based 
            Multiplex Genome Engineering in Plants.
  JOURNAL   Front Plant Sci. 2021 Jul 1;12:689937. doi: 
            10.3389/fpls.2021.689937. eCollection 2021.
  PUBMED    34276739
REFERENCE   2  (bases 1 to 3231)
  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: "Front Plant
            Sci."; date: "2021-07-1"; volume: "12:689937. doi"; pages: " 
            10.3389/fpls.2021.689937. eCollection 2021"
COMMENT     Sequence Label: pVD1 Multiplexing Dicot Edit E1_NTERM (GB2245)
FEATURES             Location/Qualifiers
     source          1..3231
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     318..337
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(556..660)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(738..1193)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(875..896)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1087..1106
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1319..1341
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1333..1350
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1334..1350
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1357..1376
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1357..1375
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1406..1415
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1410..1419
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        complement(1459..1534)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1729..1747)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1730..1747)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1765..1781)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1765..1781)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1778..1800)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1789..1805
                     /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(1813..1819,1820..1837,1838..1843))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1858..1879
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1996..2013)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2167..2755)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2247..2266)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2926..3231,1..486,487..555))
                     /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"
ORIGIN
        1 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
       61 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
      121 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
      181 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
      241 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
      301 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
      361 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
      421 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
      481 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
      541 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
      601 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
      661 cacatttccc cgaaaagtgc cacctgatgc ggtgtgaaat accgcacaga tgcgtaagga
      721 gaaaataccg catcaggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
      781 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
      841 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
      901 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
      961 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     1021 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     1081 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     1141 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtccattcg
     1201 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     1261 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
     1321 cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa
     1381 ttgggcccga cgtcgcatgc tcccggccgc catggcggcc gcgggaattc gattcccgtc
     1441 tcgctcaagc gaaaaaaagc accgactcgg tgccactttt tcaagttgat aacggactag
     1501 ccttatttta acttgctatt tctagctcta aaactgagac ggcgcgcgcc gtctctgcac
     1561 cagccgggaa tcgaacccgg gtctgtaccg tggcagggta ctattctacc actagaccac
     1621 tggtgctttg ttcaatcgag cgagacggga tcactagtga attcgcggcc gcctgcaggt
     1681 cgaccatatg ggagagctcc caacgcgttg gatgcatagc ttgagtattc tatagtgtca
     1741 cctaaatagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     1801 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     1861 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     1921 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     1981 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     2041 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     2101 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     2161 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     2221 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     2281 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     2341 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     2401 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     2461 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     2521 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     2581 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     2641 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     2701 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     2761 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     2821 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     2881 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     2941 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     3001 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     3061 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     3121 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     3181 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg t
//
