LOCUS       sequence-423191-        7315 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a DHFR-mCherry fusion protein and uses for a CRISPR gene 
            deletion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7315)
  AUTHORS   Long S, Wang Q, Sibley LD
  TITLE     Analysis of non-canonical calcium dependent protein kinases in 
            Toxoplasma gondii by targeted gene deletion using CRISPR/Cas9.
  JOURNAL   Infect Immun. 2016 Jan 11. doi: 10.1128/IAI.01173-15
  PUBMED    26755159
REFERENCE   2  (bases 1 to 7315)
  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: "Infect 
            Immun. 2016 Jan 11. doi: 10.1128/IAI.01173-15"
COMMENT     Sequence Label: pLoxP-DHFR-mCherry
FEATURES             Location/Qualifiers
     source          1..7315
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(415..448)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(3403..3421)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3671..3690
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(3976..4009)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5075..5091)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5075..5091)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        complement(5092..5110)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5118..5134
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5142..5148,5149..5166,5167..5172))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5187..5208
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5325..5342)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5496..6084)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5576..5595)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6255..7046,7047..7115))
                     /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     6878..6897
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7116..7220)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7288..7306
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccataact
      421 tcgtatagca tacattatac gaagttataa gcttcgccag gctgtaaatc ccgtgagtcg
      481 tcctcacaaa tcatcaagca ggtgtcctca gggagactgc ctgactgagt tatgctaatt
      541 cctttctact ttggcgtggt cacgggcgcg ccggatcctt aattaagtct agcatgtcat
      601 tcgattttca ccccccgcgt agttcctgtg tgtcattcgt tgtcgagaca actctgtccc
      661 gccccggtgc tgttccatat gcgtgacttt cccgcaattt tttcagactt tcaggaaaga
      721 caggctccgg aacgatctcg tccatgactg gtaaatccac gacaccgcaa tggcccccag
      781 cacctctatc tctcgtgcca ggggactaac gttgtatgcg tctgcgtctt gtctttttgc
      841 attcgctttc caaaaaagag agccatccgt tcccccgcac attcaacgcc gcgagtgcgg
      901 tttttgtctt ttttgagtgg taggacgctt ttcatgcgcg aactacgtgg acattaagtt
      961 ccattctctt tttcgacagc acgaaacctt gcattcaaac ccgcccgcgg aagatccgat
     1021 cttgctgctg ttcgcagtcc cagtagcgtc ctgtcggccg cgccgtctct gttggtgggc
     1081 agccgctaca cctgttatct gactgccgtg cgcgaaaatg acgccatttt tgggaaaatc
     1141 ggggaacttc attctttaaa agtatgcgga ggtttccttt ttcttctgtt cgtttctttt
     1201 tctcgggttt gataaccgtg ttcgatgtaa gcactttccg tctctcctcc gtgctttgtt
     1261 cgacatcgag accaggtgtg cagatccttc gcttgtcgat ccggagacgc gtgtctcgta
     1321 gaaccttttc attttaccac acggcagtgc ggagcactgc tctgagtgca gcagggacgg
     1381 gtgaagtttc gctttagtag tgcgtttctg ctctacgggg cgttgtcgtg tctgggaaga
     1441 tgcagaaacc ggtgtgtctg gtcgtcgcga tgacccccaa gaggggcatc ggcatcaaca
     1501 acggcctccc gtggccccac ttgaccacag atttcaaaca ctttcgtcgt gtgacaaaaa
     1561 cgacgcccga agaagccagt cgcctgaacg ggtggcttcc caggaaattt gcaaagacgg
     1621 gcgactctgg acttccctct ccatcagtcg gcaagagatt caacgccgtt gtcatgggac
     1681 ggaaaaactg ggaaagcatg cctcgaaagt ttagacccct cgtggacaga ttgaacatcg
     1741 tcgtttcctc ttccctcaaa gaagaagaca ttgcggcgga gaagcctcaa gctgaaggcc
     1801 agcagcgcgt ccgagtctgt gcttcactcc cagcagctct cagccttctg gaggaagagt
     1861 acaaggattc tgtcgaccag atttttgtcg tgggaggagc gggactgtac gaggcagcgc
     1921 tgtctctggg cgttgcctct cacctgtaca tcacgcgtgt agcccgcgag tttccgtgcg
     1981 acgttttctt ccctgcgttc cccggagatg acattctttc aaacaaatca actgctgcgc
     2041 aggctgcagc tcctgccgag tctgtgttcg ttcccttttg tccggagctc ggaagagaga
     2101 aggacaatga agcgacgtat cgacccatct tcatttccaa gaccttctca gacaacgggg
     2161 taccctacga ctttgtggtt ctcgagaaga gaaggaagac tgacgacgca gccactgcgg
     2221 aaccgagcaa cgcaatgagc tccttgacgt ccacgaggga gacaactccc gtgcacgggt
     2281 tgcaggctcc ttcttcggcc gcagccattg ccccggtgtt ggcgtggatg gacgaagaag
     2341 accggaaaaa acgcgagcaa aaggaactga ttcgggccgt tccgcatgtt cactttagag
     2401 gccatgaaga attccagtac cttgatctca ttgccgacat tattaacaat ggaaggacaa
     2461 tggatgaccg aacgggcgtt ggtgtcatct ccaaattcgg ctgcactatg cgctactcgc
     2521 tggatcaggc ctttccactt ctcaccacaa agcgtgtgtt ctggaaaggg gtcctcgaag
     2581 agttgctgtg gttcattcgc ggcgacacga acgcaaacca tctttctgag aagggcgtga
     2641 agatctggga caagaatgtg acacgcgagt tcctcgattc gcgcaatctc ccccaccgag
     2701 aggtcggaga catcggcccg ggctacggct tccagtggag acacttcggc gcggcataca
     2761 aagacatgca cacagactac acagggcagg gcgtcgacca gctgaagaat gtgatccaga
     2821 tgctgagaac gaatccaaca gatcgtcgca tgctcatgac tgcctggaat cctgcagcgc
     2881 tggacgaaat ggcgctgccg ccttgtcact tgttgtgcca gttctacgtg aacgaccaga
     2941 aggagctgtc gtgcatcatg tatcagcggt cgtgcgatgt cggcctcggc gtccccttca
     3001 acatcgcttc ctattcgctt ttgacgctca tggttgcaca cgtctgcaac ctaaaaccta
     3061 aggagttcat tcacttcatg gggaacacgc atgtctacac gaaccatgtc gaggctttaa
     3121 aagagcagct gcggagagaa ccgagaccgt tccccattgt gaacatcctc aacaaggaac
     3181 gcatcaagga aatcgacgat ttcaccgccg aggattttga ggtcgtgggc tacgtcccgc
     3241 acggacgaat ccagatggag atggctgtcg gtggaggtgc tagcatggcc atcatcaagg
     3301 agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg
     3361 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca
     3421 agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca
     3481 aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg
     3541 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg
     3601 actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc
     3661 cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga
     3721 tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg
     3781 gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc
     3841 ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca
     3901 tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg gacgagctgt
     3961 acaagtaagc tgagcataac ttcgtatagc atacattata cgaagttatc ggaaatacag
     4021 aagctgcccg tctctcgttt tcctctcttt tcggagggat cagggagagt gcctcgggtc
     4081 ggagagagct gacgaggggg tgccagagac ccctgtgtcc tttatcgaag aaaagggatg
     4141 actcttcatg tggcatttca cacagtctca cctcgccttg ttttcttttt gtcaatcaga
     4201 acgaaagcga gttgcgggtg acgcagatgt gcgtgtatcc actcgtgaat gcgttatcgt
     4261 tctgtatgcc gctagagtgc tggactgttg ctgtctgccc acgacagcag acaactttcc
     4321 ttctatgcac ttgcaggatg gtgcagcgca aacgacggag agaaaggagc accctctcag
     4381 tttccctacg atgtgctgtc agtttcgact cttcaccgcg aacgattggc gatacgtctc
     4441 tgttgacttg ttaggctccg accacgaagc tcccttaact aaataagccg cgacacctaa
     4501 gtgtacacca tttgcagatc gataatctgc gaccgctgaa tccgtccaga tcagtaaaac
     4561 cgcaccacct aagtgtaaac cttgtttagg tcgataaaat gctaccaacc cccacccaca
     4621 atcgagcctt gagcgtttct gcgcacgcgt tggcctacgt gacttgctga tgcctgcctc
     4681 tggccattca tgccagtcag tgcgcataaa aatgtggaca cagtcggttg acaagtgttc
     4741 tggcaggcta cagtgacacg gggaaatcgt ttctgagaga gcaccaaaac cttttagctg
     4801 tcaccatctt cttcgcttgc tacgttgtct gatttgtcgt ttttcctctc ggtgtgaagc
     4861 gtttttcgta acgtcaatcg acccaggaca ctgcttccag ctgtctgtgc cgtcctgtcg
     4921 tgaccagttt gtcgccgcga ctttatgagc tttttcctgt tgagaaagcg gttcggcggc
     4981 cgccacgtga catgcgggag aagaaaacgt gaccgaacaa atcaactggt gatctagagt
     5041 cgacctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct gcctatagtg
     5101 agtcgtatta tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     5161 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     5221 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5281 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     5341 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5401 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5461 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5521 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5581 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5641 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5701 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5761 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5821 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5881 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5941 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6001 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6061 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6121 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6181 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6241 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6301 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6361 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6421 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6481 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6541 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6601 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6661 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6721 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6781 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6841 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6901 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6961 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     7021 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     7081 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     7141 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     7201 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     7261 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtc
//
