LOCUS       sequence-117006-        3430 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tribolium U6a promoter with BsaI cloning site to insert guide RNA 
            sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3430)
  AUTHORS   Gilles AF, Schinko JB, Averof M
  TITLE     Efficient CRISPR-mediated gene targeting and transgene replacement 
            in the beetle Tribolium castaneum.
  JOURNAL   Development. 2015 Jul 9. pii: dev.125054.
  PUBMED    26160901
REFERENCE   2  (bases 1 to 3430)
  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: 
            "Development. 2015 Jul 9. pii: dev.125054."
COMMENT     Sequence Label: p(U6a-BsaI-gRNA)
FEATURES             Location/Qualifiers
     source          1..3430
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        454..529
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(586..606)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(586..604)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(625..641)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(625..641)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(638..660)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    649..665
                     /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(673..679,680..697,698..703))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    718..739
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(856..873)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1027..1615)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1107..1126)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1786..2577,2578..2646))
                     /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     2409..2428
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2647..2751)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2777..3232)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2914..2935)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3126..3145
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3359..3381
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3373..3390
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3374..3390
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3400..3419
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3400..3418
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 cgaattcccg ggattaagtg gcgcgccatt acaaggatcc cttgcaaaaa caattcgttt
       61 acgtccgtta acacgttctc gtaatccaaa aataaataat ttatggtctt tatcaaatta
      121 atcgtgtaga gaactgtatc atcaacaatt aataatttgt acatgattat tgattatgga
      181 tagtctagac taaacctttt acgaaatcaa tgactaactg ttttatttat ttatttatgt
      241 gttaatatta attgagataa ttttaagttt aatgctaata atgtaacaac ttcttattaa
      301 atagtaaaca caaacaaatt gataaaaacc agtcatattc taaaattaaa aaagttattg
      361 tggaggttac atctcactaa cgctggtcgc ccgtcgctac gttcatatac agattgttga
      421 gaagggcaag tgagagaccg agagagggtc tcagttttag agctagaaat agcaagttaa
      481 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttatcgg
      541 ccggccataa gtatcacgtg gaattccaag ctccagcttt tgttcccttt agtgagggtt
      601 aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
      661 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
      721 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
      781 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
      841 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
      901 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
      961 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     1021 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     1081 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     1141 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     1201 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     1261 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     1321 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     1381 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     1441 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     1501 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     1561 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     1621 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     1681 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     1741 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     1801 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     1861 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     1921 accgcgagat ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     1981 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     2041 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     2101 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     2161 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     2221 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     2281 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     2341 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     2401 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     2461 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     2521 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     2581 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     2641 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     2701 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     2761 ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     2821 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     2881 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     2941 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     3001 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     3061 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     3121 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     3181 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg
     3241 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     3301 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
     3361 cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc
     3421 gaattgggta
//
