LOCUS       sequence-90531-e        7635 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  replace TgUPRT with DHFR by homologous recombination.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7635)
  AUTHORS   Shen B, Brown KM, Lee TD, Sibley LD
  TITLE     Efficient Gene Disruption in Diverse Strains of Toxoplasma gondii 
            Using CRISPR/CAS9.
  JOURNAL   MBio. 2014 May 13;5(3). pii: e01114-14. doi: 10.1128/mBio.01114-14.
  PUBMED    24825012
REFERENCE   2  (bases 1 to 7635)
  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: "MBio."; 
            date: "2014-05-13"; volume: "5(3). pii: e01114-14. doi"; pages: " 
            10.1128/mBio.01114-14"
COMMENT     Sequence Label: pUPRT::DHFR-D
FEATURES             Location/Qualifiers
     source          1..7635
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(21..38)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(21..37)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(30..52)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     365..387
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(425..443)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        511..615
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(616..684,685..1476)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGSDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(834..853)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1647..2235
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2136..2155
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2362..2384
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2381..2397
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2381..2397
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    2417..2437
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    3416..3440
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(6607..6631)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(join(7628..7635,1..13))
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
ORIGIN
        1 ttatacatag ttgataattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
       61 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
      121 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc
      181 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact
      241 ctcagtgcaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
      301 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
      361 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
      421 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
      481 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
      541 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
      601 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
      661 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
      721 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
      781 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
      841 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
      901 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
      961 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     1021 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     1081 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     1141 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     1201 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     1261 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     1321 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggt cagatggtaa gccctcccgt
     1381 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     1441 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     1501 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     1561 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     1621 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     1681 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     1741 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     1801 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     1861 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     1921 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     1981 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     2041 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     2101 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     2161 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     2221 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggt cttttgctgg
     2281 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     2341 gcctttgagt gagctgatac cagcggataa caatttcaca caggaaacag ctatgaccat
     2401 gattacgcca agctatcaac tttgtataga aaagttgtcc cgctgtgcct agtatcgaaa
     2461 gctgtagagt tgccaccctg atcttctccc ttccaactct ccgcttcgac acacccccca
     2521 acgacttgtt gtgaagctct ggcccccctg tttcggttgt gacaggctct ccaggtgctc
     2581 ttgcactctc cagtccagct ggtgtcatag aaccgttatc tctagagtgc cccgaaagag
     2641 ccccctcctt ttattcgttg tttcccctgg gtacgagcta tcttgtggct ttctttgctt
     2701 ggggatgacc cttgtcgtga ccttctatgc gttctcgatg agaaaccgtc gagtcttcgt
     2761 tgcatgcagg ggtttctgcg cgaagatact ctcccacaga gccacagaaa gtggtagccg
     2821 ctgttttcca acgcagcgta cgtacctaga gatccttcca ggaaacgctg agcctccgct
     2881 gtgactgatt ttctgcacgt tggcacgtgg atatcgcgat cccctgtccc ctccactgca
     2941 tgcagtgtgg cggtaagacg agggccatat cgatcccatg gcagctgctg tgtggggtgc
     3001 cgccatacgt aggggtacga tttctggtcc tgcccaactt cccgcctggt tgattcgagc
     3061 aacttacaag gacaaggcaa ttaaccatct tgctattttt cctgttttcg tcgtcatcgg
     3121 ctgtcctagc tctcacggct acccatcgtt ttcccaagcg ctgtcaagtg atgccattgg
     3181 ttacactgga caaaggccct gcaaatacgc gtgcctccat catcgatccc cctttatcta
     3241 ctttctcgct ttccgttatc ccttctgtct gctttttcct ttctcggctc cacctcattc
     3301 ccctcgttcc ttgtagcggc cccggcttct acgttccgcc tgcgattccc gtattggtca
     3361 cgcatccaca aggcgtattc cttttttcgt cggacctgtc cacagggctt ctaaaacaag
     3421 tttgtacaaa aaagcaggct tcaagcttcg ccaggctgta aatcccgtga gtcgtcctca
     3481 caaatcatca agcaggtgtc ctcagggaga ctgcctgact gagttatgct aattcctttc
     3541 tactttggcg tggtcacggg ggcgcgccgg atccttaatt aagtctagca tgtcattcga
     3601 ttttcacccc ccgcgtagtt cctgtgtgtc attcgttgtc gagacaactc tgtcccgccc
     3661 cggtgctgtt ccatatgcgt gactttcccg caattttttc agactttcag gaaagacagg
     3721 ctccggaacg atctcgtcca tgactggtaa atccacgaca ccgcaatggc ccccagcacc
     3781 tctatctctc gtgccagggg actaacgttg tatgcgtctg cgtcttgtct ttttgcattc
     3841 gctttccaaa aaagagagcc atccgttccc ccgcacattc aacgccgcga gtgcggtttt
     3901 tgtctttttt gagtggtagg acgcttttca tgcgcgaact acgtggacat taagttccat
     3961 tctctttttc gacagcacga aaccttgcat tcaaacccgc ccgcggaaga tccgatcttg
     4021 ctgctgttcg cagtcccagt agcgtcctgt cggccgcgcc gtctctgttg gtgggcagcc
     4081 gctacacctg ttatctgact gccgtgcgcg aaaatgacgc catttttggg aaaatcgggg
     4141 aacttcattc tttaaaagta tgcggaggtt tcctttttct tctgttcgtt tctttttctc
     4201 gggtttgata accgtgttcg atgtaagcac tttccgtctc tcctccgtgc tttgttcgac
     4261 atcgagacca ggtgtgcaga tccttcgctt gtcgatccgg agacgcgtgt ctcgtagaac
     4321 cttttcattt taccacacgg cagtgcggag cactgctctg agtgcagcag ggacgggtga
     4381 agtttcgctt tagtagtgcg tttctgctct acggggcgtt gtcgtgtctg ggaagatgca
     4441 gaaaccggtg tgtctggtcg tcgcgatgac ccccaagagg ggcatcggca tcaacaacgg
     4501 cctcccgtgg ccccacttga ccacagattt caaacacttt cgtcgtgtga caaaaacgac
     4561 gcccgaagaa gccagtcgcc tgaacgggtg gcttcccagg aaatttgcaa agacgggcga
     4621 ctctggactt ccctctccat cagtcggcaa gagattcaac gccgttgtca tgggacggaa
     4681 aaactgggaa agcatgcctc gaaagtttag acccctcgtg gacagattga acatcgtcgt
     4741 ttcctcttcc ctcaaagaag aagacattgc ggcggagaag cctcaagctg aaggccagca
     4801 gcgcgtccga gtctgtgctt cactcccagc agctctcagc cttctggagg aagagtacaa
     4861 ggattctgtc gaccagattt ttgtcgtggg aggagcggga ctgtacgagg cagcgctgtc
     4921 tctgggcgtt gcctctcacc tgtacatcac gcgtgtagcc cgcgagtttc cgtgcgacgt
     4981 tttcttccct gcgttccccg gagatgacat tctttcaaac aaatcaactg ctgcgcaggc
     5041 tgcagctcct gccgagtctg tgttcgttcc cttttgtccg gagctcggaa gagagaagga
     5101 caatgaagcg acgtatcgac ccatcttcat ttccaagacc ttctcagaca acggggtacc
     5161 ctacgactcc gtggttctcg agaagagaag gaagactgac gacgcagcca ctgcggaacc
     5221 gagcaacgca atgagctcct tgacgtccac gagggagaca actcccgtgc acgggttgca
     5281 ggctccttct tcggccgcag ccattgcccc ggtgttggcg tggatggacg aagaagaccg
     5341 gaaaaaacgc gagcaaaagg aactgattcg ggccgttccg catgttcact ttagaggcca
     5401 tgaagaattc cagtaccttg atctcattgc cgacattatt aacaatggaa ggacaatgga
     5461 tgaccgaacg ggcgttggtg tcatctccaa attcggctgc actatgcgct actcgctgga
     5521 tcaggccttt ccacttctca ccacaaagcg tgtgttctgg aaaggggtcc tcgaagagtt
     5581 gctgtggttc attcgcggcg acacgaacgc aaaccatctt tctgagaagg gcgtgaagat
     5641 ctgggacaag aatgtgacac gcgagttcct cgattcgcgc aatctccccc accgagaggt
     5701 cggagacatc ggcccgggct acggcttcca gtggagacac ttcggcgcgg catacaaaga
     5761 catgcacaca gactacacag ggcagggcgt cgaccagctg aagaatgtga tccagatgct
     5821 gagaacgaat ccaacagatc gtcgcatgct catgactgcc tggaatcctg cagcgctgga
     5881 cgaaatggcg ctgccgcctt gtcacttgtt gtgccagttc tacgtgaacg accagaagga
     5941 gctgtcgtgc atcatgtatc agcggtcgtg cgatgtcggc ctcggcgtcc ccttcaacat
     6001 cgcttcctat tcgcttttga cgctcatggt tgcacacgtc tgcaacctaa aacctaagga
     6061 gttcattcac ttcatgggga acacgcatgt ctacacgaac catgtcgagg ctttaaaaga
     6121 gcagctgcgg agagaaccga gaccgttccc cattgtgaac atcctcaaca aggaacgcat
     6181 caaggaaatc gacgatttca ccgccgagga ttttgaggtc gtgggctacg tcccgcacgg
     6241 acgaatccag atggagatgg ctgtctagcg gaaatacaga agctgcccgt ctctcgtttt
     6301 cctctctttt cggagggatc agggagagtg cctcgggtcg gagagagctg acgagggggt
     6361 gccagagacc cctgtgtcct ttatcgaaga aaagggatga ctcttcatgt ggcatttcac
     6421 acagtctcac ctcgccttgt tttctttttg tcaatcagaa cgaaagcgag ttgcgggtga
     6481 cgcagatgtg cgtgtatcca ctcgtgaatg cgttatcgtt ctgtatgccg ctagagtgct
     6541 ggactgttgc tgtctgccca cgacagcaga caactttcct tctatgcact tgcaggatga
     6601 attccgaccc agctttcttg tacaaagtgg tcgtctctag tttttttgac agaccgctga
     6661 cggaatcgcg gactggaaaa agcacgcctc aagaggataa acaccatttt tatttttgtc
     6721 tcgctcccac gtccctcacc atcggcagga acacagaaaa aaagttttag ttgtgaggct
     6781 gcctggtgcg cgacaagcag ttgcgctttt ttgtgccacg gggtatatct ttggtcgcgc
     6841 tcctgtcacg actgttgtta gtcatggacc ggacgatttc ttcgtatcag gcacagagtc
     6901 ccgtacacct gtaacgggaa tgtttacgag gcggcgtgca tcccgttcct cgtccatctc
     6961 gagttttatt tccctttcct ttgttcgaga ttctgctgtc cagaacgaga cagggaagcg
     7021 aagtggtagt gccaggaagg gcgcagctta attgctcctc ttcaccttaa gtcgtttact
     7081 tgtgcccaag aagtgttctt cacgattacc ttgtgtgttt ccgccgttct ttgctttttg
     7141 ttcgcgctgc tcggaaacat ttacaacctc atatctagat gttgttgaag caatgtgaag
     7201 aatggtccac gttacattct ctaacggaat tttaacggtg tgggatggac aagcaatgca
     7261 tatgcgatgt cgaaccctcg aaaacccgta atgtgcgacc acgtcctggg tgaagcaaac
     7321 gcgtttccac atacagtcgg tatcgatgtc acgcttgcca gttcgtgtag ggaagttggc
     7381 gtgttttttt ttccacggtg gggattgaac agtgtcattg cccagaccat ggcagtacca
     7441 agtctgctct gatgaagtgc gccaccagca ttctgttttt tcgctcccac gagggtggaa
     7501 cctctagcac acaacttctc tcttctgagg ttgccgaaaa actgtctgct aatttatcga
     7561 gttatttgaa ctacgggacc aacgggcaga gactgttttc actcggatgt cgtacagttg
     7621 acgtcgacaa cttta
//
