LOCUS       sequence-178063-        8134 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for generation of AID strains using CRISPR tagging technology.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8134)
  AUTHORS   Long S, Brown KM, Drewry LL, Anthony B, Phan IQH, Sibley LD
  TITLE     Calmodulin-like proteins localized to the conoid regulate motility 
            and cell invasion by Toxoplasma gondii.
  JOURNAL   PLoS Pathog. 2017 May 5;13(5):e1006379. doi: 
            10.1371/journal.ppat.1006379.
  PUBMED    28475612
REFERENCE   2  (bases 1 to 8134)
  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: "PLoS 
            Pathog."; date: "2017-05-5"; volume: "13(5):e1006379. doi"; pages: "
            10.1371/journal.ppat.1006379"
COMMENT     Sequence Label: pLinker-AID-3xHA-DHFR-LoxP
FEATURES             Location/Qualifiers
     source          1..8134
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             692..1375
                     /codon_start=1
                     /gene="Arabidopsis thaliana AXR3"
                     /product="auxin-responsive IAA17 protein"
                     /label=auxin-inducible degron
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="MGSVELNLRETELCLGLPGGDTVAPVTGNKRGFSETVDLKLNLNN
                     EPANKEGSTTHDVVTFDSKEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSG
                     GPEAAAFVKVSMDGAPYLRKIDLRMYKSYDELSNALSNMFSSFTMGKHGGEEGMIDFMN
                     ERKLMDLVNSWDYVPSYEDKDGDWMLVGDVPWPMFVDTCKRLRLMKGSDAIGLAPRAME
                     KCKSRA"
     CDS             1412..1438
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1442..1468
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1469..1495
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     1930..1946
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1931..1947
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(1975..2008)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(4894..4927)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(5927..5945)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5966..5982)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5966..5982)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        complement(5983..6001)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6009..6025
                     /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(6033..6039,6040..6057,6058..6063))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6078..6099
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6216..6233)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6387..6975)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6467..6486)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7146..7937,7938..8006))
                     /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     7769..7788
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8007..8111)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtaccgggcc
      661 cgctagcaag ggctcgggct cgacccagct gatgggcagt gtcgagctga atctgaggga
      721 gactgagctg tgtcttggtc ttcccggtgg agatacagtg gctccggtaa ccggaaacaa
      781 gagagggttc tcagagacgg ttgatctgaa gctaaatctg aataatgagc ctgcaaacaa
      841 ggaaggatct acgactcatg acgtcgtgac ttttgattcc aaggagaaga gtgcttgtcc
      901 taaagatcca gccaaacctc cggccaaggc acaagttgtg ggatggccac cggtgagatc
      961 ataccggaag aacgtgatgg tttcctgcca aaaatcaagc ggtggcccgg aggcggcggc
     1021 gttcgtgaag gtatcaatgg acggagcacc gtacttgagg aaaatcgatt tgaggatgta
     1081 taaaagctac gatgagcttt ctaatgcttt gtccaacatg ttcagctctt ttaccatggg
     1141 caaacatgga ggagaagaag gaatgataga cttcatgaat gagaggaaat tgatggattt
     1201 ggtgaatagc tgggactatg ttccctctta tgaagacaaa gacggtgatt ggatgctcgt
     1261 cggcgacgtt ccttggccaa tgttcgtcga tacatgcaag cgtttacgtc tcatgaaagg
     1321 atcggatgcc attggtctcg ctccgagggc gatggagaag tgcaagagca gagctgggcg
     1381 cgccggctct ggcggcggcg gcggcggctc ttacccgtac gacgtcccgg actacgctgg
     1441 ctatccctat gatgtgcccg attatgcgta tccttacgat gttccagatt atgcctaacc
     1501 cgggcatatg tagaaaagtt gtaacgttag taaacgtaac acacgggtat aatagccatt
     1561 taatggcgcc gtatattttt cactccccct ctccgatagg aggaagaaag acgatccgtc
     1621 tgcgaatgta tattacatcg accggccata gtacgagaag tggtgccggt attgttctgt
     1681 ccggggggtg taccatccta ttttttgtat atgtcgttct tgtcggggag caacagccca
     1741 tcttccggct attgtctcgt atcagttgtg aaacagactg cgttttgccg aaaatgagtc
     1801 ctatgtccga tggctatttg taccgcttcg taccatttcg cttgtcagct gtaatcaacc
     1861 gaattcattt ggcgagacat gattccgatt aaattcttta ctgcgcgtgc tcggatccac
     1921 tagttctact cgaggtcgac ggtatcgatt ctagagaatt cgagctcggt acccataact
     1981 tcgtatagca tacattatac gaagttataa gcttcgccag gctgtaaatc ccgtgagtcg
     2041 tcctcacaaa tcatcaagca ggtgtcctca gggagactgc ctgactgagt tatgctaatt
     2101 cctttctact ttggcgtggt aacgggcgcg ccggatcctt aattaagtct agcatgtcat
     2161 tcgattttca ccccccgcgt agttcctgtg tgtcattcgt tgtcgagaca actctgtccc
     2221 gccccggtgc tgttccatat gcgtgacttt cccgcaattt tttcagactt tcaggaaaga
     2281 caggctccgg aacgatctcg tccatgactg gtaaatccac gacaccgcaa tggcccccag
     2341 cacctctatc tctcgtgcca ggggactaac gttgtatgcg tctgcgtctt gtctttttgc
     2401 attcgctttc caaaaaagag agccatccgt tcccccgcac attcaacgcc gcgagtgcgg
     2461 tttttgtctt ttttgagtgg taggacgctt ttcatgcgcg aactacgtgg acattaagtt
     2521 ccattctctt tttcgacagc acgaaacctt gcattcaaac ccgcccgcgg aagatccgat
     2581 cttgctgctg ttcgcagtcc cagtagcgtc ctgtcggccg cgccgtctct gttggtgggc
     2641 agccgctaca cctgttatct gactgccgtg cgcgaaaatg acgccatttt tgggaaaatc
     2701 ggggaacttc attctttaaa agtatgcgga ggtttccttt ttcttctgtt cgtttctttt
     2761 tctcgggttt gataaccgtg ttcgatgtaa gcactttccg tctctcctcc gtgctttgtt
     2821 cgacatcgag accaggtgtg cagatccttc gcttgtcgat ccggagacgc gtgtctcgta
     2881 gaaccttttc attttaccac acggcagtgc ggagcactgc tctgagtgca gcagggacgg
     2941 gtgaagtttc gctttagtag tgcgtttctg ctctacgggg cgttgtcgtg tctgggaaga
     3001 tgcagaaacc ggtgtgtctg gtcgtcgcga tgacccccaa gaggggcatc ggcatcaaca
     3061 acggcctccc gtggccccac ttgaccacag atttcaaaca ctttcgtcgt gtgacaaaaa
     3121 cgacgcccga agaagccagt cgcctgaacg ggtggcttcc caggaaattt gcaaagacgg
     3181 gcgactctgg acttccctct ccatcagtcg gcaagagatt caacgccgtt gtcatgggac
     3241 ggaaaaactg ggaaagcatg cctcgaaagt ttagacccct cgtggacaga ttgaacatcg
     3301 tcgtttcctc ttccctcaaa gaagaagaca ttgcggcgga gaagcctcaa gctgaaggcc
     3361 agcagcgcgt ccgagtctgt gcttcactcc cagcagctct cagccttctg gaggaagagt
     3421 acaaggattc tgtcgaccag atttttgtcg tgggaggagc gggactgtac gaggcagcgc
     3481 tgtctctggg cgttgcctct cacctgtaca tcacgcgtgt agcccgcgag tttccgtgcg
     3541 acgttttctt ccctgcgttc cccggagatg acattctttc aaacaaatca actgctgcgc
     3601 aggctgcagc tcctgccgag tctgtgttcg ttcccttttg tccggagctc ggaagagaga
     3661 aggacaatga agcgacgtat cgacccatct tcatttccaa gaccttctca gacaacgggg
     3721 taccctacga ctttgtggtt ctcgagaaga gaaggaagac tgacgacgca gccactgcgg
     3781 aaccgagcaa cgcaatgagc tccttgacgt ccacgaggga gacaactccc gtgcacgggt
     3841 tgcaggctcc ttcttcggcc gcagccattg ccccggtgtt ggcgtggatg gacgaagaag
     3901 accggaaaaa acgcgagcaa aaggaactga ttcgggccgt tccgcatgtt cactttagag
     3961 gccatgaaga attccagtac cttgatctca ttgccgacat tattaacaat ggaaggacaa
     4021 tggatgaccg aacgggcgtt ggtgtcatct ccaaattcgg ctgcactatg cgctactcgc
     4081 tggatcaggc ctttccactt ctcaccacaa agcgtgtgtt ctggaaaggg gtcctcgaag
     4141 agttgctgtg gttcattcgc ggcgacacga acgcaaacca tctttctgag aagggcgtga
     4201 agatctggga caagaatgtg acacgcgagt tcctcgattc gcgcaatctc ccccaccgag
     4261 aggtcggaga catcggcccg ggctacggct tccagtggag acacttcggc gcggcataca
     4321 aagacatgca cacagactac acagggcagg gcgtcgacca gctgaagaat gtgatccaga
     4381 tgctgagaac gaatccaaca gatcgtcgca tgctcatgac tgcctggaat cctgcagcgc
     4441 tggacgaaat ggcgctgccg ccttgtcact tgttgtgcca gttctacgtg aacgaccaga
     4501 aggagctgtc gtgcatcatg tatcagcggt cgtgcgatgt cggcctcggc gtccccttca
     4561 acatcgcttc ctattcgctt ttgacgctca tggttgcaca cgtctgcaac ctaaaaccta
     4621 aggagttcat tcacttcatg gggaacacgc atgtctacac gaaccatgtc gaggctttaa
     4681 aagagcagct gcggagagaa ccgagaccgt tccccattgt gaacatcctc aacaaggaac
     4741 gcatcaagga aatcgacgat ttcaccgccg aggattttga ggtcgtgggc tacgtcccgc
     4801 acggacgaat ccagatggag atggctgtcg gtgacgagct gtacaagtag atggagatgg
     4861 ctgtcggtga cgagctgtac aagtaagctg agcataactt cgtatagcat acattatacg
     4921 aagttatcgg aaatacagaa gctgcccgtc tctcgttttc ctctcttttc ggagggatca
     4981 gggagagtgc ctcgggtcgg agagagctga cgagggggtg ccagagaccc ctgtgtcctt
     5041 tatcgaagaa aagggatgac tcttcatgtg gcatttcaca cagtctcacc tcgccttgtt
     5101 ttctttttgt caatcagaac gaaagcgagt tgcgggtgac gcagatgtgc gtgtatccac
     5161 tcgtgaatgc gttatcgttc tgtatgccgc tagagtgctg gactgttgct gtctgcccac
     5221 gacagcagac aactttcctt ctatgcactt gcaggatggt gcagcgcaaa cgacggagag
     5281 aaaggagcac cctctcagtt tccctacgat gtgctgtcag tttcgactct tcaccgcgaa
     5341 cgattggcga tacgtctctg ttgacttgtt aggctccgac cacgaagctc ccttaactaa
     5401 ataagccgcg acacctaagt gtacaccatt tgcagatcga taatctgcga ccgctgaatc
     5461 cgtccagatc agtaaaaccg caccacctaa gtgtaaacct tgtttaggtc gataaaatgc
     5521 taccaacccc cacccacaat cgagccttga gcgtttctgc gcacgcgttg gcctacgtga
     5581 cttgctgatg cctgcctctg gccattcatg ccagtcagtg cgcataaaaa tgtggacaca
     5641 gtcggttgac aagtgttctg gcaggctaca gtgacacggg gaaatcgttt ctgagagagc
     5701 accaaaacct tttagctgtc accatcttct tcgcttgcta cgttgtctga tttgtcgttt
     5761 ttcctctcgg tgtgaagcgt ttttcgtaac gtcaatcgac ccaggacact gcttccagct
     5821 gtctgtgccg tcctgtcgtg accagtttgt cgccgcgact ttatgagctt tttcctgttg
     5881 agaaagcggt gcggccgcca ccgcggtgga gctccagctt ttgttccctt tagtgagggt
     5941 taatttcgag cttggcgtaa tcatggtcat agctgtttcc tgcctatagt gagtcgtatt
     6001 atgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     6061 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     6121 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     6181 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     6241 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     6301 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     6361 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     6421 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     6481 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     6541 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     6601 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     6661 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     6721 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6781 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     6841 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6901 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6961 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     7021 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     7081 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     7141 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     7201 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     7261 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     7321 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     7381 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     7441 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     7501 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     7561 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     7621 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     7681 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     7741 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     7801 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     7861 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     7921 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     7981 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     8041 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     8101 ggggttccgc gcacatttcc ccgaaaagtg ccac
//
