LOCUS       sequence-472744-        5983 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR donor plasmid for C-terminal tagging and knock-in of CFAP36 
            [A493E, K495A], and insertion of Blasticidin resistance (C. 
            reinhardtii).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5983)
  TITLE     A conserved mechanism for the retrieval of polyubiquitinated 
            proteins from cilia
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5983)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pBR25 CFAP36 [A493E, K495A]-3C-mStayGold-2Stre...
FEATURES             Location/Qualifiers
     source          1..5983
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1010..1030
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             1498..1521
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1531..2178
                     /codon_start=1
                     /product="bright and highly photostable monomeric green 
                     fluorescent protein (Ivorra-Molla et al., 2023)"
                     /label=mStayGold (E138D)
                     /note="contains the monomerizing E138D mutation"
                     /translation="ASTPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKGKYVCTSGKLP
                     MSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGSIHAKHQHFMK
                     NGTYHNIVEFTGQDFKENSPVLTGDMNVSLPNDVQHIPRDDGVECPVTLLYPLLSDKSK
                     CVEAHQNTICKPLHNQPAPDVPYHWIRKQYTQSKDDTEERDHICQSETLEAHL"
     CDS             join(2191..2214,2215..2250,2251..2274)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     primer_bind     complement(4180..4199)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4181..4199)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4209..4226)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4209..4225)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4218..4240)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4367..4822
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4454..4473)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4664..4685
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4848..4952
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4953..5021,5022..5813)
                     /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(5171..5190)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
     1021 tcactaaagg gaacaaaagc tgggctcttc tccaccatgc atcccaaccc ctccacatca
     1081 tacctatctt ctacccctcc cacttttgca gaagctcgtg ggcaagggcg acgtgaacga
     1141 cgccgccacc aagtggctac aggaggccaa gctcaagctg atcgcgcaga agcaggcgga
     1201 gcgcgagcgg gagatggtgg actacaaggc ggccagcgcc ggcaaggtgg ggaggaggcg
     1261 agggggcagg gatgggggaa aaggggaggg gagctgtgac tgggtttggt tagcggagct
     1321 gagcggctga tgtcgggcat tcacgctgct gctcgttgcc cctcccgccc gacgctgcag
     1381 cccaagccct ctggcccctc caccaccgct gccgccgcgg acgacgccaa gcgcgctgcg
     1441 ctgcgtgagg ccctggagca ggcgatgaag caaaacatca tgggagcaaa cggtggcctg
     1501 gaagttctgt tccaggggcc cggcggcggt gccagcaccc ccttcaagtt ccagctgaag
     1561 ggcaccatca acggcaagag cttcaccgtg gagggcgagg gcgagggcaa cagccacgag
     1621 ggcagccaca agggcaagta cgtgtgcacc agcggcaagc tgcccatgag ctgggccgcc
     1681 ctgggcacca gcttcggcta cggcatgaag tactacacca agtaccccag cggcctgaag
     1741 aactggttcc acgaggtgat gcccgagggc ttcacctacg acagacacat ccagtacaag
     1801 ggcgacggca gcatccacgc caagcaccag cacttcatga agaacggcac ctaccacaac
     1861 atcgtggagt tcaccggcca ggacttcaag gagaacagcc ccgtgctgac cggcgacatg
     1921 aacgtgagcc tgcccaacga cgtgcagcac atccccagag acgacggcgt ggagtgcccc
     1981 gtgaccctgc tgtaccccct gctgagcgac aagagcaagt gcgtggaggc ccaccagaac
     2041 accatctgca agcccctgca caaccagccc gcccccgacg tgccctacca ctggatcaga
     2101 aagcagtaca cccagagcaa ggacgacacc gaggagagag accacatctg ccagagcgag
     2161 accctggagg cccacctggg ttctggttct tggagtcatc cacagttcga gaagggcggg
     2221 ggctccggag gtggatcagg aggttccgcc tggtcacacc ctcaattcga gaagggtgaa
     2281 tgaggagctg ggagcatcga gagcacgggc gggggtgcgc gggggcagag cgagatgctg
     2341 acgttgccag aggtccggtt tggcaggtgg tgactgatgt gaggggtgag cgcaggtggt
     2401 gccgctggac cgaaatattg tagaggaggc atgcaggcga gcgattgcag tgtgcggacg
     2461 tgaagtgcaa taggtgctgc aggtgttttg ggttgccgtg attcgcgata gcaggactga
     2521 gtttgtgcag aggtggtttg caagcgaaat gggtttggct tattattggc ttgtgccttc
     2581 cggcgttgtt ctggcgacct ttgccaaagt gcctttggcc agtgccatgc cgcgcgcatg
     2641 tgtagtcatc ttcaacgggc attgtcatgg cattgtcact cagctaatgg tatgtcaaac
     2701 accgctgagt agttcgagga gaagaggatg tcaaacgccg ctgagtatgc acgccccagc
     2761 ctcgggcgca cagtgtcaca atcagcacta cgctgcagct cttagtactg agggtaccct
     2821 ttcttgcgct atgacacttc cgatcccatc aagcttgcat gccgggcgcg ccagaaggag
     2881 cgcagccaaa ccaggatgat gtttgatggg gtatttgagc acttgcaacc cttatccgga
     2941 agccccctgg cccacaaagg ctaggcgcca atgcaagcag ttcgcatgca gcccctggag
     3001 cggtgccctc ctgataaacc ggccaggggg cctatgttct ttactttttt acaagagaag
     3061 tcactcaaca tcttaaaatg gccaggtgag tcgacgagca agcccggcgg atcaggcagc
     3121 gtgcttgcag atttgacttg caacgcccgc attgtgtcga cgaaggcttt tggctcctct
     3181 gtcgctgtct caagcagcat ctaaccctgc gtcgccgttt ccatttgcag ctcgagcgac
     3241 gtcatggcca tgaaaacctt caacatcagc cagcaggacc tggagctggt ggaggtggcc
     3301 accgagaaga tcaccatgct gtacgaggac aacaagcacc acgtgggcgc cgccatccgc
     3361 accaagaccg gcgagatcat cagcgccgtg cacatcgagg cctacatcgg ccgcgtgacc
     3421 gtgtgcgccg aggccatcgc catcggcagc gccgtgagca acggccagaa ggacttcgac
     3481 accatcgtgg ccgtgcgcca cccctacagc gacgaggtgg accgcagcat ccgcgtggtg
     3541 agcccctgcg gcatgtgccg cgagctgatc agcgactacg cccccgactg cttcgtgctg
     3601 atcgagatga acggcaagct ggtcaagacc accatcgagg agctgatccc cctgaagtac
     3661 acccgcaact aagctttgag accatccccg ctccgtgtaa atggaggcgc tcgttgatct
     3721 gagccttgcc ccctgacgaa cggcggtgga tggaagatac tgctctcaag tgctgaagcg
     3781 gtagcttagc tccccgtttc gtgctgatca gtctttttca acacgtaaaa agcggaggag
     3841 ttttgcaatt ttgttggttg taacgatcct ccgttgattt tggcctcttt ctccatgggc
     3901 gggctgggcg tatttgaagc gggtactaat cttttgcaga agctggtggg caagggcgac
     3961 gtgaacgacg ccgccaccag aagagccaaa cgagcactta tacatacgcg actattctgc
     4021 cgctatacat aaccactcag gagctctatg tgacaatgct tccgtgacgc cgcgaggtac
     4081 ttgggtcgat atcaaagtgc atcacttctt cccgtatgcc caactttgta tagagagcca
     4141 ctgcgggatc gtcaccgtaa tctgcttgca cgtagttcgc cctatagtga gtcgtattac
     4201 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     4261 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     4321 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc
     4381 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     4441 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     4501 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     4561 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     4621 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     4681 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     4741 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     4801 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     4861 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     4921 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     4981 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5041 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5101 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5161 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     5221 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5281 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     5341 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     5401 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     5461 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     5521 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     5581 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     5641 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     5701 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     5761 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     5821 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     5881 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     5941 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttc
//
