LOCUS       sequence-165803-        6392 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid contains LIC cassette upstream of an in-frame copy of CFP 
            and HXGPRT selectable marker cassette for endogenous tagging on the 
            gene locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6392)
  AUTHORS   Huynh MH, Carruthers VB
  TITLE     Tagging of endogenous genes in a Toxoplasma gondii strain lacking 
            Ku80.
  JOURNAL   Eukaryot Cell. 2009 Apr;8(4):530-9. doi: 10.1128/EC.00358-08. Epub 
            2009 Feb 13.
  PUBMED    19218426
REFERENCE   2  (bases 1 to 6392)
  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: 
            "10.1128/EC.00358-08"; journalName: "Eukaryot Cell"; date: 
            "2009-04"; volume: "8"; issue: "4"; pages: "530-9"
COMMENT     Sequence Label: pCFP.HXGPRT
FEATURES             Location/Qualifiers
     source          1..6392
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1933..1949)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1981..2000)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1982..2000)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2007..2024)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2007..2023)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2016..2038)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2165..2620
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2252..2271)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2462..2483
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2646..2750
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2751..2819,2820..3611)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAISMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2969..2988)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3782..4370
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4524..4541
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(4694..4699,4700..4717,4718..4724)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4732..4748
                     /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)."
     primer_bind     4737..4759
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4756..4772
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4756..4772
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        4793..4811
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             join(4874..4876,4877..4879,4880..5593)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHEFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4919..4940)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5180..5199)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5527..5548
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 aagcttgatc agcacgaaac cttgcattca aacccgcccg cggaagatcc gatcttgctg
       61 ctgttcgcag tcccagtagc gtcctgtcgg ccgcgccgtc tctgttggtg ggcagccgct
      121 acacctgtta tctgactgcc gtgcgcgaaa atgacgccat ttttgggaaa atcggggaac
      181 ttcattcttt aaaagtatgc ggaggtttcc tttttcttct gttcgtttct ttttctcggg
      241 tttgataacc gtgttcgatg taagcacttt ccgtctctcc tccgtgcttt gttcgacatc
      301 gagaccaggt gtgcagatcc ttcgcttgtc gatccggaga cgcgtgtctc gtagaacctt
      361 ttcattttac cacacggcag tgcggagcac tgctctgagt gcagcaggga cgggtgaagt
      421 ttcgctttag tagtgcgttt ctgctctacg gggcgttgtc agatccaaaa tggcgtccaa
      481 acccattgaa gactacggca agggcaaggg ccgtattgag cccatgtata tccccgacaa
      541 caccttctac aacgctgatg actttcttgt gcccccccac tgcaagccct acattgacaa
      601 aatcctcctc cctggtggat tggtcaagga cagagttgag aagttggcgt atgacatcca
      661 cagaacttac ttcggcgagg agttgcacat catttgcatc ctgaaaggct ctcgcggctt
      721 cttcaacctt ctgatcgact accttgccac catacagaag tacagtggtc gtgagtccag
      781 cgtgcccccc ttcttcgagc actatgtccg cctgaagtcc taccagaacg acaacagcac
      841 aggccagctc accgtcttga gcgacgactt gtcaatcttt cgcgacaagc acgttctgat
      901 tgttgaggac atcgtcgaca ccggtttcac cctcaccgag ttcggtgagc gcctgaaagc
      961 cgtcggtccc aagtcgatga gaatcgccac cctcgtcgag aagcgcacag atcgctccaa
     1021 cagcttgaag ggcgacttcg tcggcttcag cattgaagac gtctggatcg ttggttgctg
     1081 ctacgacttc aacgagatgt tccgcgactt cgaccacgtc gccgtcctga gcgacgccgc
     1141 tcgcaaaaag ttcgagaagt aaaccctgca tagcccacag aagctgcccg tctctcgttt
     1201 tcctctcttt tcggagggat cagggagagt gcctcgggtc ggagagagct gacgaggggg
     1261 tgccagagac ccctgtgtcc tttatcgaag aaaagggatg actcttcatg tggcatttca
     1321 cacagtctca cctcgccttg ttttcttttt gtcaatcaga acgaaagcga gttgcgggtg
     1381 acgcagatgt gcgtgtatcc actcgtgaat gcgttatcgt tctgtatgcc gctagagtgc
     1441 tggactgttg ctgtctgccc acgacagcag acaactttcc ttctatgcac ttgcaggatg
     1501 gtgcagcgca aacgacggag agaaaggagc accctctcag tttccctacg atgtgctgtc
     1561 agtttcgact cttcaccgcg aacgattggc gatacgtctc tgttgacttg ttaggctccg
     1621 accacgaagc tcccttaact agataagccg cgacacctaa gtgtacacca tttgcagatc
     1681 gataatctgc gaccgctgaa tccgtccaga tcagtaaaac cgcaccacct aagtgtaaac
     1741 cttgtttagg tcgataaaat gctaccaacc cccacccaca atcgagcctt gagcgtttct
     1801 gcgcacgcgt tggcctacgt gacttgctga tgcctgcctc tggccattca tgccagtcag
     1861 tgcgcataaa aatgtggaca cagtcggttg acaagtgttc tggcaggcta cagtgacacc
     1921 gcggtggagg gggatccact agttctagag cggccgccac cgcggtggag ctccaattcg
     1981 ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg tgactgggaa
     2041 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccccttcgc cagctggcgt
     2101 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgtagcct gaatggcgaa
     2161 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2221 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2281 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2341 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     2401 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2461 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     2521 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2581 tttaacgcga attttaacaa aatattaacg tttacaattt aggtggcact tttcggggaa
     2641 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2701 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     2761 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     2821 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     2881 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     2941 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     3001 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     3061 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     3121 ccataagcat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     3181 aggagctaac cgcttttttt cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     3241 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     3301 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     3361 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     3421 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     3481 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggca
     3541 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     3601 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     3661 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     3721 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     3781 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     3841 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3901 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     3961 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4021 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4081 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4141 cctacaccga actgagatac ctacagcgtg agcattgaga aagcgccacg cttcccgaag
     4201 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4261 agcttccagg ggggaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4321 ttgagcgtcg atttttgtga tgctcgtcag gggggccgag cctatggaaa aacgccagca
     4381 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     4441 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     4501 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     4561 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     4621 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttac ctcactcatt
     4681 aggcacccca ggctttacac tttatgcttc cggctcctat gttgtgtgga attgtgagcg
     4741 gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc ggaattaacc
     4801 ctcactaaag ggaacaaaag ctggtaccct gtacttccaa tccaatttaa ttaaaattgg
     4861 aagtggagga cggatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     4921 ggtcgagctg gacggcgacg taaacggcca cgagttcagc gtgtccggcg agggcgaggg
     4981 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     5041 gccctggccc accctcgtga ccaccctgac ctggggcgtg cagtgcttca gccgctaccc
     5101 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     5161 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     5221 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     5281 catcctgggg cacaagctgg agtacaacta catcagccac aacgtctata tcaccgccga
     5341 caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcag
     5401 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     5461 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     5521 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     5581 gctgtacaag taaggcgcgc cagaagctgc ccgtctctcg ttttcctctc ttttcggagg
     5641 gatcagggag agtgcctcgg gtcggagaga gctgacgagg gggtgccaga gacccctgtg
     5701 tcctttatcg aagaaaaggg atgactcttc atgtggcatt tcacacagtc tcacctcgcc
     5761 ttgttttctt tttgtcaatc agaacgaaag cgagttgcgg gtgacgcaga tgtgcgtgta
     5821 tccactcgtg aatgcgttat cgttctgtat gccgctagag tgctggactg ttgctgtctg
     5881 cccacgacag cagacaactt tccttctatg cacttgcagg atggtgcagc gcaaacgacg
     5941 gagagaaagg agcaccctct cagtttccct acgatgtgct gtcagtttcg actcttcacc
     6001 gcgaacgatt ggcgatacgt ctctgttgac ttgttaggct ccgaccacga agctccctta
     6061 actagataag ccgcgacacc taagtgtaca ccatttgcag atcgataatc tgcgaccgct
     6121 gaatccgtcc agatcagtaa aaccgcacca cctaagtgta aaccttgttt aggtcgataa
     6181 aatgctacca acccccaccc acaatcgagc cttgagcgtt tctgcgcacg cgttggccta
     6241 cgtgacttgc tgatgcctgc ctctggccat tcatgccagt cagtgcgcat aaaaatgtgg
     6301 acacagtcgg ttgacaagtg ttctggcagg ctacagtgac accgcggtgg agggggatcc
     6361 actagttcta ctcgagggtc gacggtatcg at
//
