LOCUS       sequence-112129-        3406 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tribolium U6b 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 3406)
  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 3406)
  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(U6b-BsaI-gRNA)
FEATURES             Location/Qualifiers
     source          1..3406
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        430..505
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(555..574)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(556..574)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(584..601)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(584..600)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(593..615)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      742..1197
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(829..848)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1039..1060
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1223..1327
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1328..1396,1397..2188)
                     /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     complement(1546..1565)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2359..2947
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2848..2867
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3101..3118
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3235..3256
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3271..3276,3277..3294,3295..3301)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3309..3325
                     /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     3314..3336
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3333..3349
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3333..3349
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3368..3388
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3370..3388
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ggaattccac gtgatactta tggccggccg ataaggatcc gggtgttatt attattactt
       61 gtgacctggt gataagtgct tcaagtacta gagattgagt cactgttcaa ggattattta
      121 aaaacacaac acgcagattt atcagttacc ttatgaacaa atgaatatct atttttgttt
      181 caaaactgtt ttcattacga caaattaata tttcttcaac taaataataa aaattttaaa
      241 taagcgccat tacatacaaa tatacgaatc attacgaacg tttgtgtttt gcgatcgatt
      301 gtaaccgacc aatcaaaata aagcgggaaa tttcaaatct cttatatctc actactacgt
      361 atcggttgcc gctaactaca tatacaaggc aggtagaagc accgttcgag agaccgagag
      421 agggtctcag ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
      481 tgaaaaagtg gcaccgagtc ggtgcttttt tgtaatggcg cgccacttaa tcccgggaat
      541 tcgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac
      601 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
      661 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
      721 gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
      781 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
      841 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
      901 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
      961 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     1021 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     1081 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     1141 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     1201 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     1261 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     1321 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     1381 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     1441 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     1501 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     1561 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     1621 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     1681 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     1741 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     1801 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     1861 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     1921 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     1981 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     2041 tggatctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     2101 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     2161 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     2221 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     2281 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     2341 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     2401 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     2461 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     2521 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     2581 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     2641 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     2701 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     2761 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     2821 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     2881 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     2941 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3001 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3061 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3121 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     3181 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     3241 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
     3301 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
     3361 caagcgcgca attaaccctc actaaaggga acaaaagctg gagctt
//
