LOCUS       sequence-448026-        4864 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To make Gateway entry clone of CRISPR guide RNA under AtU6p.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4864)
  TITLE     Siddiqi August 2024 Plasmids
REFERENCE   2  (bases 1 to 4864)
  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"
COMMENT     Sequence Label: pENTR_AtU6p_BsaI_ccdb_gRNA
FEATURES             Location/Qualifiers
     source          1..4864
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1096..1101,1102..1119,1120..1126)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1180..1860
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(1246..1265)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2180..2485
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFW*I"
     primer_bind     2364..2383
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        2527..2602
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(2989..3088)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3105..3124)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3106..3124)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3129..3145)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3129..3145)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3247..3266)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3258..4067
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3325..3344)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4214..4802
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4703..4722
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctcc gcggccgcct ttttttcttc ttcttcgttc atacagtttt tttttgttta
      721 tcagcttaca ttttcttgaa ccgtagcttt cgttttcttc tttttaactt tccattcgga
      781 gtttttgtat cttgtttcat agtttgtccc aggattagaa tgattaggca tcgaaccttc
      841 aagaatttga ttgaataaaa catcttcatt cttaagatat gaagataatc ttcaaaaggc
      901 ccctgggaat ctgaaagaag agaagcaggc ccatttatat gggaaagaac aatagtattt
      961 cttatatagg cccatttaag ttgaaaacaa tcttcaaaag tcccacatcg cttagataag
     1021 aaaacgaagc tgagtttata tacagctaga gtcgaagtag tgattgagag accaagctta
     1081 ttaggcaccc caggctttac actttatgct tccggctcgt ataatgtgtg gattttgagt
     1141 taggatccgt cgagattttc aggagctaag gaagctaaaa tggagaaaaa aatcactgga
     1201 tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc atttcagtca
     1261 gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt tttaaagacc
     1321 gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc ccgcctgatg
     1381 aatgctcatc cggaattccg tatggcaatg aaagacggtg agctggtgat atgggatagt
     1441 gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc gctctggagt
     1501 gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt ggcgtgttac
     1561 ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt cgtctcagcc
     1621 aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga caacttcttc
     1681 gcccccgttt tcaccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg
     1741 gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa
     1801 ttacaacagt actgcgatga gtggcaggcg gggcgtaatc tagaggatcc ggcttactaa
     1861 aagccagata acagtatgcg tatttgcgcg ctgatttttg cggtataaga atatatactg
     1921 atatgtatac ccgaagtatg tcaaaaagag gtatgctatg aagcagcgta ttacagtgac
     1981 agttgacagc gacagctatc agttgctcaa ggcatatatg atgtcaatat ctccggtctg
     2041 gtaagcacaa ccatgcagaa tgaagcccgt cgtctgcgtg cagaacgctg gaaagcggaa
     2101 aatcaggaag ggatggctga ggtcgcccgg tttattgaaa tgaacggctc ttttgctgac
     2161 gagaacaggg gctggtgaaa tgcagtttaa ggtttacacc tataaaagag agagccgtta
     2221 tcgtctgttt gtggatgtac agagtgatat tattgacacg cccgggcgac ggatggtgat
     2281 ccccctggcc agtgcacgtc tgctgtcaga taaagtcccc cgtgaacttt acccggtggt
     2341 gcatatcggg gatgaaagct ggcgcatgat gaccaccgat atggccagtg tgccggtctc
     2401 cgttatcggg gaagaagtgg ctgatctcag ccaccgcgaa aatgacatca aaaacgccat
     2461 taacctgatg ttctggtgaa tataaatgtc aggctccctt atacacagcc agtgagctcg
     2521 gtctcagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
     2581 aaaagtggca ccgagtcggt gctttttttg gcaaaaattt tcagattttt tcttcatctg
     2641 tagatttctg ggtttttttt tccgtttcgt gaatcataag tgaagttttg gatgcaaatc
     2701 tgcgcgaaaa aagttggacc tgcaatgagc ttatttagat agctaagaca aagtgattgg
     2761 tccgttgttt cagttctgat tgtcagagag tttgtttcga gtcggcgaca ccaatgcgtt
     2821 ttgttaacca gatttcgggt aagaaatgta tcgagagttt gtttccagac ggctacatca
     2881 ttttcttatg aagggtgaaa ttagatagac caaagattga aacacaacat ttctttcaca
     2941 aaaatataat aaacttgata gcatttagga tcagccatgg gcgcgccgac ccagctttct
     3001 tgtacaaagt tggcattata agaaagcatt gcttatcaat ttgttgcaac gaacaggtca
     3061 ctatcagtca aaataaaatc attatttgcc atccagctga tatcccctat agtgagtcgt
     3121 attacatggt catagctgtt tcctggcagc tctggcccgt gtctcaaaat ctctgatgtt
     3181 acattgcaca agataaaaat atatcatcat gaacaataaa actgtctgct tacataaaca
     3241 gtaatacaag gggtgttatg agccatattc aacgggaaac gtcgaggccg cgattaaatt
     3301 ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag
     3361 gtgcgacaat ctatcgcttg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg
     3421 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg
     3481 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac
     3541 tcaccactgc gatccccgga aaaacagcat tccaggtatt agaagaatat cctgattcag
     3601 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt
     3661 gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga
     3721 ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac
     3781 aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg
     3841 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg
     3901 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg
     3961 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg
     4021 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta
     4081 attggttgta acactggcag agcattacgc tgacttgacg ggacggcgca agctcatgac
     4141 caaaatccct taacgtgagt tacgcgtcgt tccactgagc gtcagacccc gtagaaaaga
     4201 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4261 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4321 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     4381 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4441 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4501 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4561 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4621 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4681 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4741 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4801 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     4861 tgtt
//
