LOCUS       sequence-213047-        5940 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  High affinity HA Tagging of weakly expressed genes (optimized for 
            Toxoplasma gondii).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5940)
  AUTHORS   Hortua Triana MA, Marquez-Nogueras KM, Chang L, Stasic AJ, Li C, 
            Spiegel KA, Sharma A, Li ZH, Moreno SNJ
  TITLE     Tagging of Weakly Expressed Toxoplasma gondii Calcium-Related Genes 
            with High-Affinity Tags.
  JOURNAL   J Eukaryot Microbiol. 2018 Apr 19. doi: 10.1111/jeu.12626.
  PUBMED    29672999
REFERENCE   2  (bases 1 to 5940)
  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: "J Eukaryot 
            Microbiol. 2018 Apr 19. doi: 10.1111/jeu.12626."
COMMENT     Sequence Label: pLIC-SM-HA
FEATURES             Location/Qualifiers
     source          1..5940
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    38..59
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(74..79,80..97,98..104)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    112..128
                     /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     117..139
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     136..152
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     136..152
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        173..191
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             269..295
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             299..325
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             329..355
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             884..910
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             917..943
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             956..982
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             989..1015
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1226..1252
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1256..1282
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1286..1312
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     1741..1757
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1742..1758
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2273..2935
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MHEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAF
                     LKTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFS
                     SLWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVAN
                     MDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQVGA"
     primer_bind     complement(2342..2361)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     complement(3253..3269)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3301..3320)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3302..3320)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3327..3344)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3327..3343)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3336..3358)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3485..3940
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3572..3591)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3782..3803
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3966..4070
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4071..4139,4140..4931)
                     /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(4289..4308)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5102..5690
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5591..5610
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5844..5861
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
       61 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
      121 gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc gaaattaacc
      181 ctcactaaag ggaacaaaag ctggtaccct gtacttccaa tccaatttaa ttaaaattgg
      241 aagtggagga cgggaattcc ctaggatgta cccttatgat gtgcccgatt atgctggcta
      301 cccttatgat gttcctgatt acgccggata tccgtatgac gtgccagact acgcgggagg
      361 tgtgagcaag ggagaggagt tgtttaccgg cgtggtcccc atactggtgg agttggatgg
      421 cgacgttaat ggacataaat tctcggtccg cggcgaggga gagggagacg ccaccaatgg
      481 caagctgacg cttaagttca tttgcacgac tggaaaattg cctgtcccct ggccgaccct
      541 ggtcaccaca ctgggcggag gagtgcagtg cttctcccgt tacccagacc acatgaagca
      601 gcacgatttc ttcaagagcg caatgccgga gggatacgtg caagaacgga ctatttcctt
      661 caaggatgac ggaacctata agacccgtgc ggaggtcaaa ttcgagggtg acaccctggt
      721 gaaccgaatt gaactcaaag gaatcgattt caaggaggat ggaaatattc tgggtcacaa
      781 gctggaatac aacttcaaca gccataatgt gtacattacg gctgataagc agaagaacgg
      841 cattaaggcc aatttcaaga tccgccacaa cgttgagggt ggatatccct acgacgtgcc
      901 cgattatgcg ggcggatatc cgtatgatgt tccagattat gctggtggtg gaggctatcc
      961 ctatgatgtc cccgactacg ccggaggtta cccatacgac gtgcccgact acgctggagg
     1021 cgatggcagc gtgcagctgg cagatcatta tcaacagaat acccccatag gtgatggccc
     1081 cgttctgctt ccagataatc actacctttc cacccagagc gtgctttcga aagacccgaa
     1141 cgaaaagcgt gatcacatgg tcctgctgga gtttgtgacc gcggctggaa tcaccctggg
     1201 tatggacgaa ctctacaagg gtggctaccc ctacgatgtg ccggattacg ctggatatcc
     1261 gtatgacgta ccggactatg ccggttaccc gtacgatgtc ccggactacg cttaacatat
     1321 gtagaaaagt tgtaacgtta gtaaacgtaa cacacgggta taatagccat ttaatggcgc
     1381 cgtatatttt tcactccccc tctccgatag gaggaagaaa gacgatccgt ctgcgaatgt
     1441 atattacatc gaccggccat agtacgagaa gtggtgccgg tattgttctg tccggggggt
     1501 gtaccatcct attttttgta tatgtcgttc ttgtcgggga gcaacagccc atcttccggc
     1561 tattgtctcg tatcagttgt gaaacagact gcgttttgcc gaaaatgagt cctatgtccg
     1621 atggctattt gtaccgcttc gtaccatttc gcttgtcagc tgtaatcaac cgaattcatt
     1681 tggcgagaca tgattccgat taaattcttt actgcgcgtg ctcggatcca ctagttctac
     1741 tcgaggtcga cggtatcgat aagcttgatg gcgatgcatg tcccgcgttc gtgaaattct
     1801 ctgcatcagc ggagtgatca ggaatcatcg tctcagcggg atgacgttgc ggagcaggcc
     1861 ggctcgcggt gggcagtcag atgccgaagg cgtaactcag gacggcttgc gctcatcgca
     1921 gaacaggggt ggtgcctgca ttgggtgcgg ttggtgatcc tggttggacc ggtggagatg
     1981 cgcgcgcacg aaggggatgt gtcagaaaca ttttgtttgt tctctgtgaa cttttagatg
     2041 tgttaaaggc ggcgaatatt agcagagagt cctccttgtt ggattctctc ttgaatttcg
     2101 ccctttcctt ctctttgcga gtctcgtaga gaacaagcac tcgttcgccg tccctgacga
     2161 cgcaacccgc gcagaagaca tccaccaaac ggtgttacac aatcaccttg tgtgaagttc
     2221 ttgcggaaaa ctactcgttg gcattttttc ttgaattccc tttttcgaca aaatgcatga
     2281 gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt
     2341 tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac
     2401 ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat
     2461 tcttgcccgc ctgatgaatg ctcatccgga attccgtatg gcaatgaaag acggtgagct
     2521 ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt
     2581 ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca
     2641 agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat
     2701 gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa
     2761 tatggacaac ttcttcgccc ccgttttcac catgggcaaa tattatacgc aaggcgacaa
     2821 ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg
     2881 cagaatgctt aatgaattac aacagtactg cgatgagtgg caggtcgggg cgtaattaat
     2941 caccgttgtg ctcacttctc aaatcgacaa aggaaacaca cttcgtgcag catgtgcccc
     3001 attataaaga aactgagttg ttccgttgtg gcttgcaggt gtcacatcca caaaaaccgg
     3061 ccgactctaa ataggagtgt ttcgcagcaa gcagcgaaag tttatgactg ggtccgaatc
     3121 tctgaacgga tgtgtggcgg acctggctga tgttgatcgc cgtcgacaca cgcgccacat
     3181 gggtcaatac acaagacagc tatcagttgt tttagtcgaa ccggttaaca caattcttgc
     3241 ccccccgagg gggatccact agttctagag cggccgccac cgcggtggag ctccaattcg
     3301 ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg tgactgggaa
     3361 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     3421 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     3481 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     3541 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     3601 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     3661 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     3721 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     3781 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     3841 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     3901 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     3961 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     4021 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     4081 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     4141 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     4201 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     4261 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     4321 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     4381 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     4441 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     4501 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     4561 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     4621 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     4681 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     4741 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     4801 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     4861 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     4921 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     4981 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     5041 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5101 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5161 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5221 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     5281 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5341 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5401 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5461 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5521 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5581 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5641 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5701 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5761 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     5821 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     5881 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
//
