LOCUS       sequence-151095-        6284 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Allows for expression of an sgRNA in Toxoplasma gondii and confers 
            resistance to pyrimethamine.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6284)
  AUTHORS   Sidik SM, Huet D, Ganesan SM, Huynh MH, Wang T, Nasamu AS, Thiru P, 
            Saeij JP, Carruthers VB, Niles JC, Lourido S
  TITLE     A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential 
            Apicomplexan Genes.
  JOURNAL   Cell. 2016 Sep 8;166(6):1423-1435.e12. doi: 
            10.1016/j.cell.2016.08.019. Epub 2016 Sep 2.
  PUBMED    27594426
REFERENCE   2  (bases 1 to 6284)
  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"; doi: 
            "10.1016/j.cell.2016.08.019"; journalName: "Cell"; date: "2016-09-8-
            8"; volume: "166"; issue: "6"; pages: "1423-1435.e12"
COMMENT     Sequence Label: pU6-DHFR
FEATURES             Location/Qualifiers
     source          1..6284
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(3056..3072)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3056..3072)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     rep_origin      3157..3612
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3244..3263)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3454..3475
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3629..3648)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3748..3770
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3808..3826)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3894..3998
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3999..4067,4068..4859)
                     /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(4217..4236)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5030..5618
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5519..5538
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_RNA        6192..6267
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 ctagaggtac catgcatcta gcatgtcatt cgattttcac cccccgcgta gttcctgtgt
       61 gtcattcgtt gtcgagacaa ctctgtcccg ccccggtgct gttccatatg cgtgactttc
      121 ccgcaatttt ttcagacttt caggaaagac aggctccgga acgatctcgt ccatgactgg
      181 taaatccacg acaccgcaat ggcccccagc acctctatct ctcgtgccag gggactaacg
      241 ttgtatgcgt ctgcgtcttg tctttttgca ttcgctttcc aaaaaagaga gccatccgtt
      301 cccccgcaca ttcaacgccg cgagtgcggt ttttgtcttt tttgagtggt aggacgcttt
      361 tcatgcgcga actacgtgga cattaagttc cattctcttt ttcgacagca cgaaaccttg
      421 cattcaaacc cgcccgcgga agatccgatc ttgctgctgt tcgcagtccc agtagcgtcc
      481 tgtcggccgc gccgtctctg ttggtgggca gccgctacac ctgttatctg actgccgtgc
      541 gcgaaaatga cgccattttt gggaaaatcg gggaacttca ttctttaaaa gtatgcggag
      601 gtttcctttt tcttctgttc gtttcttttt ctcgggtttg ataaccgtgt tcgatgtaag
      661 cactttccgt ctctcctccg tgctttgttc gacatcgaga gcaggtgtgc agatccttcg
      721 cttgtcgatc cggagacgcg tgtctcgtag aaccttttca ttttaccaca cggcagtgcg
      781 gagcactgct ctgagtgcag cagggacggg tgaagtttcg ctttagtagt gcgtttctgc
      841 tctacggggc gttgtcgtgt ctgggaagat gcagaaaccg gtgtgtctgg tcgtcgcgat
      901 gacccccaag aggggcatcg gcatcaacaa cggcctcccg tggccccact tgaccacaga
      961 tttcaaacac tttcgtcgtg tgacaaaaac gacgcccgaa gaagccagtc gcctgaacgg
     1021 gtggcttccc aggaaatttg caaagacggg cgactctgga cttccctctc catcagtcgg
     1081 caagagattc aacgccgttg tcatgggacg gaaaaactgg gaaagcatgc ctcgaaagtt
     1141 tagacccctc gtggacagat tgaacatcgt cgtttcctct tccctcaaag aagaagacat
     1201 tgcggcggag aagcctcaag ctgaaggcca gcagcgcgtc cgagtctgtg cttcactccc
     1261 agcagctctc agccttctgg aggaagagta caaggattct gtcgaccaga tttttgtcgt
     1321 gggaggagcg ggactgtacg aggcagcgct gtctctgggc gttgcctctc acctgtacat
     1381 cacgcgtgta gcccgcgagt ttccgtgcga cgttttcttc cctgcgttcc ccggagatga
     1441 cattctttca aacaaatcaa ctgctgcgca ggctgcagct cctgccgagt ctgtgttcgt
     1501 tcccttttgt ccggagctcg gaagagagaa ggacaatgaa gcgacgtatc gacccatctt
     1561 catttccaag accttctcag acaacggggt accctacgac tttgtggttc tcgagaagag
     1621 aaggaagact gacgacgcag ccactgcgga accgagcaac gcaatgagct ccttgacgtc
     1681 cacgagggag acaactcccg tgcacgggtt gcaggctcct tcttcggccg cagccattgc
     1741 cccggtgttg gcgtggatgg acgaagaaga ccggaaaaaa cgcgagcaaa aggaactgat
     1801 tcgggccgtt ccgcatgttc actttagagg ccatgaagaa ttccagtacc ttgatctcat
     1861 tgccgacatt attaacaatg gaaggacaat ggatgaccga acgggcgttg gtgtcatctc
     1921 caaattcggc tgcactatgc gctactcgct ggatcaggcc tttccacttc tcaccacaaa
     1981 gcgtgtgttc tggaaagggg tcctcgaaga gttgctgtgg ttcattcgcg gcgacacgaa
     2041 cgcaaaccat ctttctgaga agggcgtgaa gatctgggac aagaatgtga cacgcgagtt
     2101 cctcgattcg cgcaatctcc cccaccgaga ggtcggagac atcggcccgg gctacggctt
     2161 ccagtggaga cacttcggcg cggcatacaa agacatgcac acagactaca cagggcaggg
     2221 cgtcgaccag ctgaagaatg tgatccagat gctgagaacg aatccaacag atcgtcgcat
     2281 gctcatgact gcctggaatc ctgcagcgct ggacgaaatg gcgctgccgc cttgtcactt
     2341 gttgtgccag ttctacgtga acgaccagaa ggagctgtcg tgcatcatgt atcagcggtc
     2401 gtgcgatgtc ggcctcggcg tccccttcaa catcgcttcc tattcgcttt tgacgctcat
     2461 ggttgcacac gtctgcaacc taaaacctaa ggagttcatt cacttcatgg ggaacacgca
     2521 tgtctacacg aaccatgtcg aggctttaaa agagcagctg cggagagaac cgaggccgtt
     2581 ccccattgtg aacatcctca acaaggaacg catcaaggaa atcgacgatt tcaccgccga
     2641 ggattttgag gtcgtgggct acgtcccgca cggacgaatc cagatggaga tggctgtcta
     2701 gcggaaatac agaagctgcc cgtctctcgt tttcctctct tttcggaggg atcagggaga
     2761 gtgcctcggg tcggagagag ctgacgaggg ggtgccagag agccctgtgt cctttatcga
     2821 agaaaaggga tgactcttca tgtggcattt cacacagtct cacctcgcct tgttttcttt
     2881 ttgtcaatca gaacgaaagc gagttgcggg tgacgcagat gtgcgtgtat ccactcgtga
     2941 atgcgttatc gttctgtatg ccgctagagt gctggactgt tgctgtctgc ccacgacagc
     3001 agacaacttt ccttctatgc acttgcagga tggaattcct gcagcccggg ggatcgatcc
     3061 actagttcta gagccctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     3121 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     3181 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3241 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3301 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3361 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3421 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3481 cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct
     3541 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     3601 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3661 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3721 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3781 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     3841 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     3901 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3961 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     4021 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     4081 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     4141 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     4201 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     4261 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     4321 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     4381 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     4441 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     4501 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     4561 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4621 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     4681 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg
     4741 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     4801 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4861 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4921 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4981 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     5041 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     5101 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     5161 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     5221 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     5281 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     5341 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     5401 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     5461 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     5521 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5581 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5641 tacggttcct ggccttttgc tggccttttg ctcacatggg atgagacaaa gtgcgcgagt
     5701 tgaaatcgtc gtggggacga tttcaccgcg gccacatgtt ggagacactg agggcacacg
     5761 ggaaacgcga aagatttcaa attaacgtac ccaaacgcga aagcttgcgc agcatacact
     5821 cgaagcgaac atcccgaacc atcgagaggc ggagagcgat aagtctttca cgctgcgaag
     5881 tgttgcgacg gctgcgccgc tgcactgtga attgggcgcc aatattgcat cctaggcctg
     5941 acgcgcctcc tgcagaacgc gagacactgg gatatgtaga gccaaggggg aaaccttcga
     6001 actctcgaat gtcttctctg acaagaatca tatttccatc agttctgtca gattttcaaa
     6061 tggcgacctg cagaggcctg cttcctccct gtgcgctctt cgaaggggct ttctgtcgcg
     6121 cagggtcacc tcgtccccga agggggtgtt tgccttctgg taaatgggga tgtcaagttg
     6181 agaccggtct cgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
     6241 cttgaaaaag tggcaccgag tcggtgcttt ttttttcttt ttct
//
