LOCUS       sequence-165796-        8047 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid contains LIC cassette upstream of an in-frame copy of YFP 
            and DHFR-TS 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 8047)
  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 8047)
  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: pYFP.LIC.DHFR
FEATURES             Location/Qualifiers
     source          1..8047
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(3583..3599)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3631..3650)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3632..3650)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3657..3674)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3657..3673)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3666..3688)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3815..4270
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3902..3921)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4112..4133
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4296..4400
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4401..4469,4470..5261)
                     /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(4619..4638)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5432..6020
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6174..6191
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(6344..6349,6350..6367,6368..6374)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6382..6398
                     /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     6387..6409
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6406..6422
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6406..6422
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        6443..6461
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             join(6530..6532,6533..6535,6536..7249)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGVDELYS"
     primer_bind     complement(6575..6596)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6836..6855)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7183..7204
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     8028..8044
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     8029..8045
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 aagcttcgcc aggctgtaaa tcccgtgagt cgtcctcaca aatcatcaag caggtgtcct
       61 cagggagact gcctgactga gttatgctaa ttcctttcta ctttggcgtg gtcacgtgta
      121 accatatccg aatcatttct ctagccctac gaacaggtaa gagcgctagg gatgtccgtg
      181 gagtagtgtg cttactcgat aatattcagt tgggactacc agcgaggcgc tcgctttgct
      241 cacgcaatgc ctgagacagt tgcagaatga atggtaaccg acaaacgcgt tcatatgcgt
      301 tttcaaactt agtagacgcg tactgtctga aactggcggt cacaggcacc agataacgcc
      361 cttggcatcg gcatgtctcg tacagaggtc cgtatgtagt gccacgactt ctaaatccgg
      421 cgacaggctg gtcttttgtc ttaccacgta ttagcccgcg tgcgatttct cggagcgcac
      481 ctgttcaaca ctagaaaacg gagtttcctg atcgagaagc caccaccttt ccagaagttg
      541 aacgctagca tgtcattcga ttttcacccc ccgcgtagtt cctgtgtgtc attcgttgtc
      601 gagacaactc tgtcccgccc cggtgctgtt ccatatgcgt gactttcccg caattttttc
      661 agactttcag gaaagacagg ctccggaacg atctcgtcca tgactggtaa atccacgaca
      721 ccgcaatggc ccccagcacc tctatctctc gtgccagggg actaacgttg tatgcgtctg
      781 cgtcttgtct ttttgcattc gctttccaaa aaagagagcc atccgttccc ccgcacattc
      841 aacgccgcga gtgcggtttt tgtctttttt gagtggtagg acgcttttca tgcgcgaact
      901 acgtggacat taagttccat tctctttttc gacagcacga aaccttgcat tcaaacccgc
      961 ccgcggaaga tccgatcttg ctgctgttcg cagtcccagt agcgtcctgt cggccgcgcc
     1021 gtctctgttg gtgggcagcc gctacacctg ttatctgact gccgtgcgcg aaaatgacgc
     1081 catttttggg aaaatcgggg aacttcattc tttaaaagta tgcggaggtt tcctttttct
     1141 tctgttcgtt tctttttctc gggtttgata accgtgttcg atgtaagcac tttccgtctc
     1201 tcctccgtgc tttgttcgac atcgagacca ggtgtgcaga tccttcgctt gtcgatccgg
     1261 agacgcgtgt ctcgtagaac cttttcattt taccacacgg cagtgcggag cactgctctg
     1321 agtgcagcag ggacgggtga agtttcgctt tagtagtgcg tttctgctct acggggcgtt
     1381 gtcgtgtctg ggaagatgca gaaaccggtg tgtctggtcg tcgcgatgac ccccaagagg
     1441 ggcatcggca tcaacaacgg cctcccgtgg ccccacttga ccacagattt caaacacttt
     1501 cgtcgtgtga caaaaacgac gcccgaagaa gccagtcgcc tgaacgggtg gcttcccagg
     1561 aaatttgcaa agacgggcga ctctggactt ccctctccat cagtcggcaa gagattcaac
     1621 gccgttgtca tgggacggaa aaactgggaa agcatgcctc gaaagtttag acccctcgtg
     1681 gacagattga acatcgtcgt ttcctcttcc ctcaaagaag aagacattgc ggcggagaag
     1741 cctcaagctg aaggccagca gcgcgtccga gtctgtgctt cactcccagc agctctcagc
     1801 cttctggagg aagagtacaa ggattctgtc gaccagattt ttgtcgtggg aggagcggga
     1861 ctgtacgagg cagcgctgtc tctgggcgtt gcctctcacc tgtacatcac gcgtgtagcc
     1921 cgcgagtttc cgtgcgacgt tttcttccct gcgttccccg gagatgacat tctttcaaac
     1981 aaatcaactg ctgcgcaggc tgcagctcct gccgagtctg tgttcgttcc cttttgtccg
     2041 gagctcggaa gagagaagga caatgaagcg acgtatcgac ccatcttcat ttccaagacc
     2101 ttctcagaca acggggtacc ctacgactcc gtggttctcg agaagagaag gaagactgac
     2161 gacgcagcca ctgcggaacc gagcaacgca atgagctcct tgacgtccac gagggagaca
     2221 actcccgtgc acgggttgca ggctccttct tcggccgcag ccattgcccc ggtgttggcg
     2281 tggatggacg aagaagaccg gaaaaaacgc gagcaaaagg aactgattcg ggccgttccg
     2341 catgttcact ttagaggcca tgaagaattc cagtaccttg atctcattgc cgacattatt
     2401 aacaatggaa ggacaatgga tgaccgaacg ggcgttggtg tcatctccaa attcggctgc
     2461 actatgcgct actcgctgga tcaggccttt ccacttctca ccacaaagcg tgtgttctgg
     2521 aaaggggtcc tcgaagagtt gctgtggttc attcgcggcg acacgaacgc aaaccatctt
     2581 tctgagaagg gcgtgaagat ctgggacaag aatgtgacac gcgagttcct cgattcgcgc
     2641 aatctccccc accgagaggt cggagacatc ggcccgggct acggcttcca gtggagacac
     2701 ttcggcgcgg catacaaaga catgcacaca gactacacag ggcagggcgt cgaccagctg
     2761 aagaatgtga tccagatgct gagaacgaat ccaacagatc gtcgcatgct catgactgcc
     2821 tggaatcctg cagcgctgga cgaaatggcg ctgccgcctt gtcacttgtt gtgccagttc
     2881 tacgtgaacg accagaagga gctgtcgtgc atcatgtatc agcggtcgtg cgatgtcggc
     2941 ctcggcgtcc ccttcaacat cgcttcctat tcgcttttga cgctcatggt tgcacacgtc
     3001 tgcaacctaa aacctaagga gttcattcac ttcatgggga acacgcatgt ctacacgaac
     3061 catgtcgagg ctttaaaaga gcagctgcgg agagaaccga gaccgttccc cattgtgaac
     3121 atcctcaaca aggaacgcat caaggaaatc gacgatttca ccgccgagga ttttgaggtc
     3181 gtgggctacg tcccgcacgg acgaatccag atggagatgg ctgtctagcg gaaatacaga
     3241 agctgcccgt ctctcgtttt cctctctttt cggagggatc agggagagtg cctcgggtcg
     3301 gagagagctg acgagggggt gccagagacc cctgtgtcct ttatcgaaga aaagggatga
     3361 ctcttcatgt ggcatttcac acagtctcac ctcgccttgt tttctttttg tcaatcagaa
     3421 cgaaagcgag ttgcgggtga cgcagatgtg cgtgtatcca ctcgtgaatg cgttatcgtt
     3481 ctgtatgccg ctagagtgct ggactgttgc tgtctgccca cgacagcaga caactttcct
     3541 tctatgcact tgcaggatgg aattcctgca gcccggggga tcgatccact agttctagag
     3601 cggccgccac cgcggtggag ctccaattcg ccctatagtg agtcgtatta caattcactg
     3661 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     3721 gcagcacatc cccccttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     3781 tcccaacagt tgcgtagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     3841 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3901 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3961 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     4021 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     4081 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     4141 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     4201 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     4261 tttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4321 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     4381 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     4441 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     4501 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     4561 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     4621 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     4681 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     4741 gcatgacagt aagagaatta tgcagtgctg ccataagcat gagtgataac actgcggcca
     4801 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttt cacaacatgg
     4861 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     4921 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     4981 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     5041 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     5101 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     5161 cccgtatcgt agttatctac acgacgggca gtcaggcaac tatggatgaa cgaaatagac
     5221 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     5281 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     5341 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     5401 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     5461 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     5521 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     5581 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     5641 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5701 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5761 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     5821 agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5881 gcagggtcgg aacaggagag cgcacgaggg agcttccagg ggggaacgcc tggtatcttt
     5941 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6001 gggggccgag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     6061 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     6121 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     6181 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     6241 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     6301 acgcaattaa tgtgagttac ctcactcatt aggcacccca ggctttacac tttatgcttc
     6361 cggctcctat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     6421 accatgatta cgccaagctc ggaattaacc ctcactaaag ggaacaaaag ctggtaccct
     6481 gtacttccaa tccaatttaa ttaaaattgg aagtggagga cgggaattca tggtgagcaa
     6541 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     6601 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     6661 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     6721 cttcggctac ggcctgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt
     6781 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     6841 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     6901 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     6961 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     7021 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     7081 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcta
     7141 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     7201 cgtgaccgcc gccgggatca ctctcggcgt ggacgagctg tacagttaag gcgcgccaga
     7261 agctgcccgt ctctcgtttt cctctctttt cggagggatc agggagagtg cctcgggtcg
     7321 gagagagctg acgagggggt gccagagacc cctgtgtcct ttatcgaaga aaagggatga
     7381 ctcttcatgt ggcatttcac acagtctcac ctcgccttgt tttctttttg tcaatcagaa
     7441 cgaaagcgag ttgcgggtga cgcagatgtg cgtgtatcca ctcgtgaatg cgttatcgtt
     7501 ctgtatgccg ctagagtgct ggactgttgc tgtctgccca cgacagcaga caactttcct
     7561 tctatgcact tgcaggatgg tgcagcgcaa acgacggaga gaaaggagca ccctctcagt
     7621 ttccctacga tgtgctgtca gtttcgactc ttcaccgcga acgattggcg atacgtctct
     7681 gttgacttgt taggctccga ccacgaagct cccttaacta gataagccgc gacacctaag
     7741 tgtacaccat ttgcagatcg ataatctgcg accgctgaat ccgtccagat cagtaaaacc
     7801 gcaccaccta agtgtaaacc ttgtttaggt cgataaaatg ctaccaaccc ccacccacaa
     7861 tcgagccttg agcgtttctg cgcacgcgtt ggcctacgtg acttgctgat gcctgcctct
     7921 ggccattcat gccagtcagt gcgcataaaa atgtggacac agtcggttga caagtgttct
     7981 ggcaggctac agtgacaccg cggtggaggg ggatccacta gttctactcg aggtcgacgg
     8041 tatcgat
//
