LOCUS       sequence-335786-        8529 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR interference. Construct for expressing catalytically inactive
            Cas9 (dCas9) from Streptococcus thermophilus (CRISPR1 locus) in 
            Toxoplasma gondii. Suitable for generating stable cell lines..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8529)
  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 8529)
  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: pTUB1-Sth.dCas9-P2A-CAT-T2A-TagBFP
FEATURES             Location/Qualifiers
     source          1..8529
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     51..70
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     304..321
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     438..454
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     438..454
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(1265..1966)
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             complement(1973..2026)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(2045..2704)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MHEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAF
                     LKTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFS
                     SLWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVAN
                     MDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2616..2635
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             complement(2714..2770)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(2780..2827)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(6224..6244)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(6251..6316)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(6885..6901)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6935..6953)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7021..7125
                     /gene="bla"
                     /label=AmpR promoter
     CDS             7126..7920
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             7126..7920
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(7180..7199)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7790..7809
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      join(8091..8529,1..150)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
       61 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
      121 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
      181 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
      241 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
      301 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
      361 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
      421 agtgagcgca acgcaatcag gaaacagcta tgaccactgt gagatgatta aacaagttac
      481 aagcttatcg ataccgtcga ccgtccagac aattggtgtc actgtagcct gccagaacac
      541 ttgtcaaccg actgtgtcca catttttatg cgcactgact ggcatgaatg gccagaggca
      601 ggcatcagca agtcacgtag gccaacgcgt gcgcagaaac gctcaaggct cgattgtggg
      661 tgggggttgg tagcatttta tcgacctaaa caaggtttac acttaggtgg tgcggtttta
      721 ctgatctgga cggattcagc ggtcgcagat tatcgatctg caaatggtgt acacttaggt
      781 gtcgcggctt atctagttaa gggagcttcg tggtcggagc ctaacaagtc aacagagacg
      841 tatcgccaat cgttcgcggt gaagagtcga aactgacagc acatcgtagg gaaactgaga
      901 gggtgctcct ttctctccgt cgtttgcgct gcaccatcct gcaagtgcat agaaggaaag
      961 ttgtctgctg tcgtgggcag acagcaacag tccagcactc tagcggcata cagaacgata
     1021 acgcattcac gagtggatac acgcacatct gcgtcacccg caactcgctt tcgttctgat
     1081 tgacaaaaag aaaacaaggc gaggtgagac tgtgtgaaat gccacatgaa gagtcatccc
     1141 ttttcttcga taaaggacac aggggtctct ggcaccccct cgtcagctct ctccgacccg
     1201 aggcactctc cctgatccct ccgaaaagag aggaaaacga gagacgggca gcttctgtgg
     1261 atccttaatt aagcttgtgc cccagtttgc tagggaggtc gcagtatctg gccactgcca
     1321 cctcgtgctg ctcgacgtag gtctcgttgt tggcctcctt gattctttcc agtctgtagt
     1381 ccacatagta gacgccaggc atcttgaggt tcttagcggg tttcttggat ctatatgtgg
     1441 tcttgatgtt tgcgatcaga tggctcccgc ccacgagctt cagggccatg tcgtttctgc
     1501 cttccaggcc gccgtcagcg gggtacagcg tctcggtgaa ggcctcccag ccgagtgttt
     1561 tcttctgcat cacagggccg ttggatgtga agttcacccc tctgatcttg acgttgtaga
     1621 tgaggcagcc gtcctggagg ctggtgtcct gggtagcggt cagcacgccc ccgtcttcgt
     1681 atgtggtgac tctctcccat gtgaagccct cagggaagga ctgcttgaag aagtcgggga
     1741 tgccctgggt gtggttgatg aaggtcttgc tgccgtagag gaagctagta gccaggatgt
     1801 cgaaggcgaa ggggagaggg ccgccctcga ccaccttgat tctcatggtc tgggtgccct
     1861 cgtagggctt gccttcgccc tcggatgtgc acttgaagtg atggttgtcc acggtgccct
     1921 ccatgtacag cttcatgtgc atgttctcct taatcagctc gctcatacta gtagggccgg
     1981 gattctcctc cacgtcaccg catgttagaa gacttcctct gccctctccg cttccctgca
     2041 ggccagcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga
     2101 catggaagcc atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt
     2161 cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg tccatattgg
     2221 ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat
     2281 tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg
     2341 aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg
     2401 tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct
     2461 caccgtcttt cattgccata cggaattccg gatgagcatt catcaggcgg gcaagaatgt
     2521 gaataaatgc cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa
     2581 tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat
     2641 gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcat
     2701 gcattccgct agcaggtcca gggttctcct ccacgtctcc agcctgcttc agcaggctga
     2761 agttagtagc tccgcttccc tttttctttt ttgcctggcc ggcctttttc gtggccgccg
     2821 gccttttctc gaggaagtcc agcttcggct tgtcgccctc gttcttgatg atgtgctggt
     2881 tgcccagcac gtcggtgcgc accttgtaga tcgagatgtt cgacttgccc aggcccttct
     2941 tgcactggcc cgagttggcc acgttgccca gcaccttgat cagggcctcg ccgccctcga
     3001 acttctgctt gtcgtacggc ttcagctcca cgtagtgctt ctgcttcggc atggtgcgcg
     3061 acaggaagcg gaacagctgc tgctccttgg tctcggtgtc cttcaccagc agcaggtcgt
     3121 tcttgtacag ggtgaacttg aactccgagt ccgagtccac gccctccttc ttcttgatgt
     3181 cgttgtactt ctcctgcgag atcttgtagg tgccggtgcc cttgtcgaac tgcaggtcgg
     3241 cgtacttcag gcccaggatc tcgtacttgc cggtggtctt gttgaagtac acgtcggcgc
     3301 gccacggcga caccgactgc agcaccacct tgttgttcga gtccttcggg gtgatgtcga
     3361 tgtggttgcc cagcttcgag tcgtagtact tcagcgactt gatctccggg ccgttgccct
     3421 tcttcgagta cttgcggatg tagccgtgct cctccttgta cttcaggaac gggttgcacg
     3481 gcacctcctt gcccttgtcg ttgatctgct tgttcgggta gttctccagg atcggctcga
     3541 tcaccttctc gaaggtctgc gggtcgtggc ggtacatcag gaacttcgac ttgtccttct
     3601 tgtagatctt catgaaggcg tcgtagccgt cctgggtgta gatgtccttg atcttgccca
     3661 gcacgtaggt ctcgtcggcc ttgtccttgc ccaccttggc ctggcgggtg gcgtagatgg
     3721 tggcgtccga gatcttgcgg ttgaacttcg agtccacctg gtacgagaac aggatcgagt
     3781 cctcgaactc cttcgacttc agggtgtcca cgaagtgctg gtacggggcc ttgaacaccg
     3841 actccttgta ctcgtcgtcc gagatcagct cgccggtctc gatgtccagc agctggtcct
     3901 ccgagtacga caccagggtg ttcttctgct tcttccacag gttcagctgc gacgaggcgg
     3961 cgatgatcag ggcgtccacg gcgtggtggt ggtaggtgtc gcgggtcttc tcgatgcccc
     4021 agtggcggcg cagctgcgag gtgaactggc cgcgcaccac cgacaccttg gtgtcgatct
     4081 tgtgggcgcg gaagtgctcc tgcagggcgt tcagcaccac gcgcgaggcg tagcgggtgt
     4141 ccaccaggtt gcgctcgatg aacttcttgc gcacgtcgaa cttcgagatg tcctcctcgg
     4201 tcagcaggta ctccttcttc ttgttcgaca gggtcttcga ctcgcgcacg aaggccttca
     4261 gctcgcggaa cgaccaggcg tcgtccatcg agtccagggc ctggtacggg gtgcgctggc
     4321 ccttctcctg gttggcggtg gcgtacacca gcaccttgtt ggccagcgag tcgtcgaagg
     4381 tgatcgacag cggcaggatg gcgtccacct cgaactggtt cgagttgttg atcaggtcgt
     4441 ggatcgagat ggtcttgccg gtgtacaggc agcgctcgcc ctgctggtgc cacaggcgga
     4501 tcttggtggc cagctgcttg tggccgtgga acaccgagtg cggcagctcg gccttgccgt
     4561 tgtactggtt ggcggccttc agcatggcgg cgtccttctc gtccttgttg gccttctgga
     4621 tcttctggat ggccttcttc tcgtcgtcct cgttggtctc gcgggccatc tcgatcacga
     4681 tgttgtcgaa gtcgccgtac tccttgatgg cggcgttcac gatcttgatg gcctggcgca
     4741 ccgacttggc caccaccggg ttgtagatct cctcggtcag cagcttctcg tcgatgtact
     4801 tggtcttgtt cgacgacgag gtggtcttct gcttgcccag gcgggtcagg atggtcatct
     4861 gctcctccga ggtctcgtac agctccggga tcagctccat catcagcttc accgagaagt
     4921 tgtgccagcc cttgccgaag atcgacgagt tggccttgcg gaactgcacc agctcgtcca
     4981 cctgcttctg cgagaacgag ccgtcggcga actcgtgctc cagggcctcc tggatgccct
     5041 cgcgctcggt gttcagggtc agcacgtagg ccagcttgtc cagggtctcg cggtccatct
     5101 gctcgatgtc cagggtctcc agggtcttca tcttgcggta ggcctcgaag gtgtggatct
     5161 cggccttgcc cgacttgtcg atgcggtagc ccttgatgtc ggccacgtcg cacgacagca
     5221 gcttggcgat gtacttgaac agcttggccg ggcccatggc cttctcgttc ttcacgtagt
     5281 tgatgatctg gttcttctgc tccttcgaca gcttcttggt ctcggtcggc acggtcaggt
     5341 tgttcaggtc gttcagcagg ttgaactcct gggcggtgta cgaggccttg gcggcgcgga
     5401 actcgtccgg gtagaaggtg cacttgccga tcaggatgcc gaagatgttg tccagggtct
     5461 cgcccgaggt gcggtagcgg ccgtagtcgg tgcgcgactt ctcgttgccc gggccgtggt
     5521 agtacttgcg cttgccggtc aggatctcca ggtagcggtt gatgaactcg tcggtgatct
     5581 gcgggttgaa ctcctgctgg gtctgcagga tgcgcagggc ctccgagcgg taggccgagg
     5641 tcgggaacac gttgatcagg cggtgcttct tgccgtcctt ctccacggtg aagtcgccgc
     5701 gcagctggcc gtaggtctgg tagcgctcca gctggatctg gcccggggtc ttggtctcca
     5761 gctgcttcga gttctccttc acgatctggg cgtagtcgcc caccgacgag ttgccgtcgt
     5821 ccgaggcgtc gtccaggtac gagatgccgc ggtgcttcac catgttcttc agggcgatga
     5881 acagctcctc gttcgacagc tcgtcggtca ggcccttcac gcgcagctgg tacgggttca
     5941 ggttgatcga gatcttggtg aagtcggtga tcaggcccga ctcctcgaac aggcggttca
     6001 ggcgcacgcg gcggtgcttc ttgcggcggg ccaggcggcg gccctggcgg ttggtgcggc
     6061 gcaccaggtt gttctcggcc tgggcggccg ggaagatgcg cgagttcttg tggatgatct
     6121 cgccggtcac cttgttcagg atgcccacgc ccaccgagcc gatgccgatg gccaggccca
     6181 gcaccaggtc cgacatatgg gctgctggga ctccgtggat accgaccttc cgcttcttct
     6241 ttggggccat cttatcgtca tcgtctttgt aatcaatatc atgatccttg tagtctccgt
     6301 cgtggtcctt atagtccgcc attttgtcga aaaagggaat tcaagaaaaa atgccaacga
     6361 gtagttttcc gcaagaactt cacacaaggt gattgtgtaa caccgtttgg tggatgtctt
     6421 ctgcgcgggt tgcgtcgtca gggacggcga acgagtgctt gttctctacg agactcgcaa
     6481 agagaaggaa agggcgaaat tcaagagaga atccaacaag gaggactctc tgctaatatt
     6541 cgccgccttt aacacatcta aaagttcaca gagaacaaac aaaatgtttc tgacacatcc
     6601 ccttcgtgcg cgcgcatctc caccggtcca accaggatca ccaaccgcac ccaatgcagg
     6661 caccacccct gttctgcgat gagcgcaagc cgtcctgagt tacgccttcg gcatctgact
     6721 gcccaccgcg agccggcctg ctccgcaacg tcatcccgct gagacgatga ttcctgatca
     6781 ctccgctgat gcagagaatt tcacgaacgc gggacatgca tgcggccgcg tacccaattc
     6841 gcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgactggc cgtcgtttta
     6901 ccaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     6961 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     7021 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     7081 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgat tgaacaagat
     7141 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     7201 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     7261 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg
     7321 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     7381 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     7441 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     7501 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     7561 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     7621 gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc
     7681 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     7741 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     7801 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     7861 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     7921 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     7981 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     8041 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     8101 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     8161 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     8221 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     8281 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     8341 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     8401 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     8461 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     8521 gacaggtat
//
