LOCUS       sequence-338002-        7202 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for generating Tetrahymena thermophila somatic knockout 
            strains for Q22YU3 with Paromomycin selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7202)
  AUTHORS   Mali GR, Ali FA, Lau CK, Begum F, Boulanger J, Howe JD, Chen ZA, 
            Rappsilber J, Skehel M, Carter AP
  TITLE     Shulin packages axonemal outer dynein arms for ciliary targeting.
  JOURNAL   Science. 2021 Feb 26;371(6532):910-916. doi: 
            10.1126/science.abe0526.
  PUBMED    33632841
REFERENCE   2  (bases 1 to 7202)
  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: "10.1126/science"; 
            journalName: "Science"; date: "2021-02-26- 26"; volume: "371"; 
            issue: "6532"; pages: "910-916"
COMMENT     Sequence Label: pBluescript-Q22YU3-KO
FEATURES             Location/Qualifiers
     source          1..7202
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..125
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(126..194,195..986)
                     /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(344..363)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1157..1745
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1646..1665
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1899..1916
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2033..2054
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2069..2074,2075..2092,2093..2099)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2107..2123
                     /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     2112..2134
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2131..2147
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2131..2147
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2168..2186
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             3206..3271
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3275..3295
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4996..5790
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MASLDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5050..5069)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5660..5679
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6517..6533)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6518..6534)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(6558..6577)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6559..6577)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6584..6601)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6584..6600)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6593..6615)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6742..7197
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6829..6848)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7039..7060
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
       61 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
      121 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
      181 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
      241 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
      301 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
      361 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
      421 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
      481 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
      541 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
      601 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
      661 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
      721 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
      781 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
      841 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
      901 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
      961 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     1021 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     1081 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1141 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1201 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1261 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     1321 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1381 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1441 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1501 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     1561 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1621 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     1681 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     1741 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     1801 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     1861 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     1921 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     1981 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     2041 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     2101 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     2161 agctcgaaat taaccctcac taaagggaac aaaagctgga gctccaccgc ggtggcggcc
     2221 gctctagagc atgcgctagc ggatccgtca ttaaaaaatt aattattaaa aaattaaatc
     2281 cgaggcttga gaaaataatg aatcataata ttaaatctat cctaaaaatt atccctctta
     2341 ctttttttag gttatttaat ttgatctttc ttaaaacagc tttgattaca gaaaatagaa
     2401 gagaagaatt tatagctagt aaaaaacaaa attaattaaa agcctttatt taaatttagt
     2461 aacaaacaat cagccaaatt cttattaatc aaatctaccc agacagatcc ttaataaact
     2521 ctttctgaat taacattttt ataatttcgt tcaccctctt agtcatcagg aaaatttaat
     2581 ttaaaatcaa attaacaatt gattctaaaa tacatacaat taattgcttt ttaagatggc
     2641 taactttacc taactaaaaa aacttattaa gtatcttacc aaatattagc atcacttatt
     2701 tgccttttgc tgattgagca tttgaattaa atttctcaaa atagatttac ctttaaaata
     2761 aaattaaatg gtatttttaa caattaattt tctttgctga aaataaaatc aagaataaaa
     2821 accaatcaat gatcgaatga gctaagaata gcttagaaag ccttttaaaa ttaaaatcat
     2881 agtatgcagc ttaagtatct tgagaaccac tttacaatag ttattatcat aataagatta
     2941 agaaaagaag tttaactctc tattatttat aacacccaac caaacctttt attataaaaa
     3001 atcaacatta taattaaata taatctctaa agcatcgctt gatattattg tctgtaaatt
     3061 tgtgtatatt tattaacctt cctattgtgc atatgagttt ttatagtttt taatttatat
     3121 tcataaataa atattactaa aatcattttt ttttttcata aacaaaaatt atttccaaaa
     3181 ataaattaat tttataacaa tcatagatta caaagatcat gatggtgatt acaaagatca
     3241 cgatattgat tacaaagatg atgatgataa agctgaaaat ctttattttc aaggtgcttc
     3301 tgctcaacat gatgaagctg ttgataacaa atttaataag gaacaacaaa atgcttttta
     3361 cgaaatttta catcttccta atttaaatga agaacaaaga aatgctttca ttcaatcatt
     3421 aaaagatgat ccttcacaat ctgctaattt attagctgaa gctaaaaaat taaatgatgc
     3481 tcaagctcct aaagttgata ataaattcaa caaagaacaa caaaacgctt tttatgaaat
     3541 tcttcattta cctaacctta atgaagaaca aagaaacgct ttcattcaat ctttaaagga
     3601 tgatccttct caatctgcta atttacttgc tgaagctaaa aagttaaatg gtgctcaagc
     3661 tcctaaggtt gatgctaata ctagttgagc gaactgaatc ggtcagctaa accaaccaat
     3721 caacataata aactttatta tttttactta agcatcttac tgttgttgta atagtagaga
     3781 aagaaatacc caattaactt cattcacata acattaatat ctataaacat cttttttctc
     3841 acatatatac aactctctaa atcaacaaat aactttttaa aaataatgga tatatattaa
     3901 caaaataata tatctctttt tacaaaatag ttcttatata aatacgtatt ctgcactcac
     3961 ccgcattttt cacaacaaaa acataccaaa aaaattctta cttctacatg tttcctttct
     4021 tattattaca aaattatttt ataaatagca tacaaaaata aatacaataa aaaaataaac
     4081 aaaatccaga tcccccgggc tgcaggaatt cgatatacaa tttatttcta aaaaatattt
     4141 aaaaataaaa aataataagg gttttgaata actcctttaa tttaaataca catttttaaa
     4201 ttttttttag ctctttaaat attcataaaa ataaaaaata actaactaaa aataaataaa
     4261 aagataataa tgattaaagg tataatactg aataagaaaa aacataatag agtacttatt
     4321 ttttatatca ctatttttaa tatcttgaaa gcaaaacttt tttatatatc ttaaaatata
     4381 ttgtatcgtt tattcaatta ttttctttaa atttcaaata tattgataaa aaagatgaca
     4441 tgttttttaa agaaaacatg aaatataaaa tagataaata tcaattattt tatttattaa
     4501 atatataagc tgctcaaaac atagctcatt catcaattat aatatgtgaa tcattaattt
     4561 tcaaaatatt actcattatt taggctatca tttatttttt attttcaatt atccgtttct
     4621 attatatttt aatattaagt tgtgattctt gaattttgtg tcatgaatta tttgtaaatc
     4681 tttttatttc tgataaaaaa tataaattga ttgactcatg atttaaatca tgagtcaacc
     4741 taactaattt tcaaaattct tctattctaa aatatagatg tgattcttga atctctcttg
     4801 aatataaagt aattttttat atttctgata taattcttag ctacgtgatt cacgatttat
     4861 gcaatgatcc atataaaata atgtaaatag tgtatatata tatattcgtc ttttttattc
     4921 tttatataat ttaaaaaaat taaaaaaatt taataaagct ctaataaaat aaataataat
     4981 actaaactta aaataatggc aagcttggat ggattgcacg caggttctcc ggccgcttgg
     5041 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     5101 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     5161 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     5221 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     5281 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     5341 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     5401 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     5461 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     5521 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     5581 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     5641 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     5701 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     5761 ttctatcgcc ttcttgacga gttcttctga gatccttaaa ttaaaaattc aatatatatt
     5821 tacaaacttt catataaaat aaatatatta tataaaatta atttttagtg tattatatta
     5881 acattaaagc accaaaaaaa cgtgttaata tactactata aaatataatt tattccaaat
     5941 tgactaaaat cattatttta caactcattt gtatatatat tttatgtcaa ttattttttt
     6001 taactttcta aaaaaaaaaa ttcctcttca catacatgtt agctcttaaa aatttgtctg
     6061 caaatccaat aataatattt ttttttgcca ttaaattttc aaatttttac tggaaaaatg
     6121 caaaattaat aattaaaata aaattagaaa aagatgaatt tattgaagaa tatttaaagt
     6181 aagaagaagt aagaataaac caaattaatg aacagttgta ataagaatgg gaaacatgga
     6241 aataagttta tgataagatt catttagaca agtgattaat tataaatatt aatatttaaa
     6301 ctgttcaata tttgtctcaa gaacttgtta ttttatacct ttaagatttt aatcatttaa
     6361 tttattaaaa gcttccaatg aaataagtct taatttaatt ttatcttgca tgaaatattt
     6421 aattcaaata aaaggagttt aatttcattt aaatgtttat ttagattact taatttatag
     6481 agtgcccggg ggatctgaat tcgatatcaa gcttatcgat accgtcgacc tcgagggggg
     6541 gcccggtacc caattcgccc tatagtgagt cgtattacaa ttcactggcc gtcgttttac
     6601 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     6661 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     6721 gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     6781 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     6841 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     6901 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     6961 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     7021 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     7081 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     7141 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     7201 tg
//
