LOCUS       sequence-335785-        6286 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR interference. Recipient construct for cloning 20-nt spacers 
            into the sgRNA scaffold compatible with Streptococcus thermophilus 
            dCas9 (CRISPR1 system) via BsaI restriction sites..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6286)
  AUTHORS   Markus BM, Boydston EA, Lourido S
  TITLE     CRISPR-Mediated Transcriptional Repression in Toxoplasma gondii.
  JOURNAL   mSphere. 2021 Oct 13:e0047421. doi: 10.1128/mSphere.00474-21.
  PUBMED    34643425
REFERENCE   2  (bases 1 to 6286)
  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: "mSphere. 
            2021 Oct 13:e0047421. doi: 10.1128/mSphere.00474-21."
COMMENT     Sequence Label: pU6-Universal.Sth.dCas9
FEATURES             Location/Qualifiers
     source          1..6286
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(890..1478)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(970..989)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1649..2440,2441..2509))
                     /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     2272..2291
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2510..2614)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2682..2700
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2738..2760)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2860..2879
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2896..3351)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3033..3054)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3245..3264
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3436..3452
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3436..3452
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
ORIGIN
        1 gagatagagg tgctgggggc cattgcggtg tcgtggattt accagtcatg gacgagatcg
       61 ttccggagcc tgtctttcct gaaagtctga aaaaattgcg ggaaagtcac gcatatggaa
      121 cagcaccggg gcgggacaga gttgtctcga caacgaatga cacacaggaa ctacgcgggg
      181 ggtgaaaatc gaatgacatg ctagatgcat ggtacctcta gagaaaaaga aaaaaaaaca
      241 ccctgccata aaatgacagg gtgttgattt cggcatgaag ccttatcttt gtagcttctt
      301 tcgagtacaa aaactgagac cggtctctaa cttgacatcc ccatttacca gaaggcaaac
      361 acccccttcg gggacgaggt gaccctgcgc gacagaaagc cccttcgaag agcgcacagg
      421 gaggaagcag gcctctgcag gtcgccattt gaaaatctga cagaactgat ggaaatatga
      481 ttcttgtcag agaagacatt cgagagttcg aaggtttccc ccttggctct acatatccca
      541 gtgtctcgcg ttctgcagga ggcgcgtcag gcctaggatg caatattggc gcccaattca
      601 cagtgcagcg gcgcagccgt cgcaacactt cgcagcgtga aagacttatc gctctccgcc
      661 tctcgatggt tcgggatgtt cgcttcgagt gtatgctgcg caagctttcg cgtttgggta
      721 cgttaatttg aaatctttcg cgtttcccgt gtgccctcag tgtctccaac atgtggccgc
      781 ggtgaaatcg tccccacgac gatttcaact cgcgcacttt gtctcatccc atgtgagcaa
      841 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
      901 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
      961 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     1021 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     1081 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     1141 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     1201 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     1261 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     1321 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     1381 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     1441 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     1501 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     1561 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     1621 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     1681 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     1741 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcgg cttccacgct
     1801 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     1861 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     1921 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     1981 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     2041 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     2101 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     2161 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     2221 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     2281 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     2341 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     2401 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     2461 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     2521 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     2581 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     2641 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     2701 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     2761 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     2821 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
     2881 tgagagtgca ccataaaatt gtaaacgtta atattttgtt aaaattcgcg ttaaattttt
     2941 gttaaatcag ctcatttttt aaccaataga ccgaaatcgg caaaatccct tataaatcaa
     3001 aagaatagcc cgagatagag ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     3061 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     3121 gtgaaccatc acccaaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     3181 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     3241 aggaagggaa gaaagcgaaa ggagcgggcg ctaaggcgct ggcaagtgta gcggtcacgc
     3301 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tactatggtt
     3361 gctttgacgt atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     3421 gcgcccctgc agggctctag aactagtgga tcgatccccc gggctgcagg aattccatcc
     3481 tgcaagtgca tagaaggaaa gttgtctgct gtcgtgggca gacagcaaca gtccagcact
     3541 ctagcggcat acagaacgat aacgcattca cgagtggata cacgcacatc tgcgtcaccc
     3601 gcaactcgct ttcgttctga ttgacaaaaa gaaaacaagg cgaggtgaga ctgtgtgaaa
     3661 tgccacatga agagtcatcc cttttcttcg ataaaggaca cagggctctc tggcaccccc
     3721 tcgtcagctc tctccgaccc gaggcactct ccctgatccc tccgaaaaga gaggaaaacg
     3781 agagacgggc agcttctgta tttccgctag acagccatct ccatctggat tcgtccgtgc
     3841 gggacgtagc ccacgacctc aaaatcctcg gcggtgaaat cgtcgatttc cttgatgcgt
     3901 tccttgttga ggatgttcac aatggggaac ggcctcggtt ctctccgcag ctgctctttt
     3961 aaagcctcga catggttcgt gtagacatgc gtgttcccca tgaagtgaat gaactcctta
     4021 ggttttaggt tgcagacgtg tgcaaccatg agcgtcaaaa gcgaatagga agcgatgttg
     4081 aaggggacgc cgaggccgac atcgcacgac cgctgataca tgatgcacga cagctccttc
     4141 tggtcgttca cgtagaactg gcacaacaag tgacaaggcg gcagcgccat ttcgtccagc
     4201 gctgcaggat tccaggcagt catgagcatg cgacgatctg ttggattcgt tctcagcatc
     4261 tggatcacat tcttcagctg gtcgacgccc tgccctgtgt agtctgtgtg catgtctttg
     4321 tatgccgcgc cgaagtgtct ccactggaag ccgtagcccg ggccgatgtc tccgacctct
     4381 cggtggggga gattgcgcga atcgaggaac tcgcgtgtca cattcttgtc ccagatcttc
     4441 acgcccttct cagaaagatg gtttgcgttc gtgtcgccgc gaatgaacca cagcaactct
     4501 tcgaggaccc ctttccagaa cacacgcttt gtggtgagaa gtggaaaggc ctgatccagc
     4561 gagtagcgca tagtgcagcc gaatttggag atgacaccaa cgcccgttcg gtcatccatt
     4621 gtccttccat tgttaataat gtcggcaatg agatcaaggt actggaattc ttcatggcct
     4681 ctaaagtgaa catgcggaac ggcccgaatc agttcctttt gctcgcgttt tttccggtct
     4741 tcttcgtcca tccacgccaa caccggggca atggctgcgg ccgaagaagg agcctgcaac
     4801 ccgtgcacgg gagttgtctc cctcgtggac gtcaaggagc tcattgcgtt gctcggttcc
     4861 gcagtggctg cgtcgtcagt cttccttctc ttctcgagaa ccacaaagtc gtagggtacc
     4921 ccgttgtctg agaaggtctt ggaaatgaag atgggtcgat acgtcgcttc attgtccttc
     4981 tctcttccga gctccggaca aaagggaacg aacacagact cggcaggagc tgcagcctgc
     5041 gcagcagttg atttgtttga aagaatgtca tctccgggga acgcagggaa gaaaacgtcg
     5101 cacggaaact cgcgggctac acgcgtgatg tacaggtgag aggcaacgcc cagagacagc
     5161 gctgcctcgt acagtcccgc tcctcccacg acaaaaatct ggtcgacaga atccttgtac
     5221 tcttcctcca gaaggctgag agctgctggg agtgaagcac agactcggac gcgctgctgg
     5281 ccttcagctt gaggcttctc cgccgcaatg tcttcttctt tgagggaaga ggaaacgacg
     5341 atgttcaatc tgtccacgag gggtctaaac tttcgaggca tgctttccca gtttttccgt
     5401 cccatgacaa cggcgttgaa tctcttgccg actgatggag agggaagtcc agagtcgccc
     5461 gtctttgcaa atttcctggg aagccacccg ttcaggcgac tggcttcttc gggcgtcgtt
     5521 tttgtcacac gacgaaagtg tttgaaatct gtggtcaagt ggggccacgg gaggccgttg
     5581 ttgatgccga tgcccctctt gggggtcatc gcgacgacca gacacaccgg tttctgcatc
     5641 ttcccagaca cgacaacgcc ccgtagagca gaaacgcact actaaagcga aacttcaccc
     5701 gtccctgctg cactcagagc agtgctccgc actgccgtgt ggtaaaatga aaaggttcta
     5761 cgagacacgc gtctccggat cgacaagcga aggatctgca cacctgctct cgatgtcgaa
     5821 caaagcacgg aggagagacg gaaagtgctt acatcgaaca cggttatcaa acccgagaaa
     5881 aagaaacgaa cagaagaaaa aggaaacctc cgcatacttt taaagaatga agttccccga
     5941 ttttcccaaa aatggcgtca ttttcgcgca cggcagtcag ataacaggtg tagcggctgc
     6001 ccaccaacag agacggcgcg gccgacagga cgctactggg actgcgaaca gcagcaagat
     6061 cggatcttcc gcgggcgggt ttgaatgcaa ggtttcgtgc tgtcgaaaaa gagaatggaa
     6121 cttaatgtcc acgtagttcg cgcatgaaaa gcgtcctacc actcaaaaaa gacaaaaacc
     6181 gcactcgcgg cgttgaatgt gcgggggaac ggatggctct cttttttgga aagcgaatgc
     6241 aaaaagacaa gacgcagacg catacaacgt tagtcccctg gcacga
//
