LOCUS       sequence-473431-        5995 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR donor plasmid for C-terminal tagging and knock-in of CFAP36 
            [L438E, K442E], and insertion of Blasticidin resistance (C. 
            reinhardtii).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5995)
  TITLE     A conserved mechanism for the retrieval of polyubiquitinated 
            proteins from cilia
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5995)
  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            "Unpublished"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 [L438E, K442E]-3C-mStayGold-2Stre...
FEATURES             Location/Qualifiers
     source          1..5995
                     /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(4221..4238)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4221..4237)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4230..4252)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4379..4834
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4466..4485)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4676..4697
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4860..4964
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4965..5033,5034..5825)
                     /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(5183..5202)
                     /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 aagtgggagc aggaggccga 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 ccctggcgca gaagatgaag 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 gctggatgga atcggcggtc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     4261 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     4321 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac
     4381 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     4441 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4501 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4561 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     4621 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4681 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     4741 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4801 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     4861 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4921 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     4981 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     5041 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     5101 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     5161 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     5221 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     5281 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     5341 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     5401 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     5461 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     5521 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5581 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5641 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     5701 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     5761 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     5821 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     5881 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5941 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttc
//
