LOCUS       sequence-337998-        7261 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for generating Tetrahymena thermophila somatic knockout 
            strains for Q22MS1 with Paromomycin selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7261)
  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 7261)
  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-Q22MS1-KO
FEATURES             Location/Qualifiers
     source          1..7261
                     /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             2667..2732
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2736..2756
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4457..5251
                     /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(4511..4530)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5121..5140
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6576..6592)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6577..6593)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(6617..6636)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6618..6636)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6643..6660)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6643..6659)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6652..6674)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6801..7256
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6888..6907)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7098..7119
                     /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 ggatcctaaa ttttgtatca aaaatagaaa aattaaatct
     2281 ttttcaagat taaaatatat tttcttgtta tttatttaat aaaaattaaa atattatttt
     2341 atttattttt gaaaaataga atgtttttaa aacttattag aattaattat taataaaaat
     2401 tatttatttt taagtaaagt gaaataaaca atttatcagt tttacatttt tagggttatt
     2461 aacagttaac actgagatag gttgttttaa aaattttaat tatagtcttt gaacttagaa
     2521 attatttttt ttgaaaatca tttttttaaa aataaaaaat aattatttat taattaatta
     2581 ttgaatataa aaaagaagcg caaattcaac atttaaaata ttcagcaact taaaaaatta
     2641 tatatttttt taatttttaa atatatgatt acaaagatca tgatggtgat tacaaagatc
     2701 acgatattga ttacaaagat gatgatgata aagctgaaaa tctttatttt caaggtgctt
     2761 ctgctcaaca tgatgaagct gttgataaca aatttaataa ggaacaacaa aatgcttttt
     2821 acgaaatttt acatcttcct aatttaaatg aagaacaaag aaatgctttc attcaatcat
     2881 taaaagatga tccttcacaa tctgctaatt tattagctga agctaaaaaa ttaaatgatg
     2941 ctcaagctcc taaagttgat aataaattca acaaagaaca acaaaacgct ttttatgaaa
     3001 ttcttcattt acctaacctt aatgaagaac aaagaaacgc tttcattcaa tctttaaagg
     3061 atgatccttc tcaatctgct aatttacttg ctgaagctaa aaagttaaat ggtgctcaag
     3121 ctcctaaggt tgatgctaat actagttgag cgaactgaat cggtcagcta aaccaaccaa
     3181 tcaacataat aaactttatt atttttactt aagcatctta ctgttgttgt aatagtagag
     3241 aaagaaatac ccaattaact tcattcacat aacattaata tctataaaca tcttttttct
     3301 cacatatata caactctcta aatcaacaaa taacttttta aaaataatgg atatatatta
     3361 acaaaataat atatctcttt ttacaaaata gttcttatat aaatacgtat tctgcactca
     3421 cccgcatttt tcacaacaaa aacataccaa aaaaattctt acttctacat gtttcctttc
     3481 ttattattac aaaattattt tataaatagc atacaaaaat aaatacaata aaaaaataaa
     3541 caaaatccag atcccccggg ctgcaggaat tcgatataca atttatttct aaaaaatatt
     3601 taaaaataaa aaataataag ggttttgaat aactccttta atttaaatac acatttttaa
     3661 atttttttta gctctttaaa tattcataaa aataaaaaat aactaactaa aaataaataa
     3721 aaagataata atgattaaag gtataatact gaataagaaa aaacataata gagtacttat
     3781 tttttatatc actattttta atatcttgaa agcaaaactt ttttatatat cttaaaatat
     3841 attgtatcgt ttattcaatt attttcttta aatttcaaat atattgataa aaaagatgac
     3901 atgtttttta aagaaaacat gaaatataaa atagataaat atcaattatt ttatttatta
     3961 aatatataag ctgctcaaaa catagctcat tcatcaatta taatatgtga atcattaatt
     4021 ttcaaaatat tactcattat ttaggctatc atttattttt tattttcaat tatccgtttc
     4081 tattatattt taatattaag ttgtgattct tgaattttgt gtcatgaatt atttgtaaat
     4141 ctttttattt ctgataaaaa atataaattg attgactcat gatttaaatc atgagtcaac
     4201 ctaactaatt ttcaaaattc ttctattcta aaatatagat gtgattcttg aatctctctt
     4261 gaatataaag taatttttta tatttctgat ataattctta gctacgtgat tcacgattta
     4321 tgcaatgatc catataaaat aatgtaaata gtgtatatat atatattcgt cttttttatt
     4381 ctttatataa tttaaaaaaa ttaaaaaaat ttaataaagc tctaataaaa taaataataa
     4441 tactaaactt aaaataatgg caagcttgga tggattgcac gcaggttctc cggccgcttg
     4501 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     4561 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     4621 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     4681 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     4741 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     4801 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     4861 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     4921 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     4981 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     5041 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     5101 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     5161 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     5221 cttctatcgc cttcttgacg agttcttctg agatccttaa attaaaaatt caatatatat
     5281 ttacaaactt tcatataaaa taaatatatt atataaaatt aatttttagt gtattatatt
     5341 aacattaaag caccaaaaaa acgtgttaat atactactat aaaatataat ttattccaaa
     5401 ttgactaaaa tcattatttt acaactcatt tgtatatata ttttatgtca attatttttt
     5461 ttaactttct aaaaaaaaaa attcctcttc acatacatgt tagctcttaa aaatttgtct
     5521 gcaaatccaa taataatatt tttttttgcc attaaatttt caaattttta ctggaaaaat
     5581 gcaaatctct tataggagtc aaatatctga aaattttgaa tttataaatc aattataatt
     5641 ataactaaaa attatttatc ctgatattat aacattttat ccaaaaaaaa atatgaattt
     5701 acttttttta tgtaaattgt atatttacta taaatacaaa tttatattat aaaaatatat
     5761 tttaataatt tatgaagatt aacattgtaa tcaatagtct ttagaaaaga tgggtttcta
     5821 cattttttga tttaattcaa aatcaaaatg aaaaactata taaaatttat ttgtaatttt
     5881 atatctaaaa ttaaaaaaat aatatgtatt tttgataaaa ttttaatgac ttaatttatt
     5941 ttaaatttta agaaattaga attttgtaaa aattaagtgt ttttgttcaa atattgctca
     6001 gaattgattt ataaaattta aaatatgatc attattaata tgtaaggatt ttcaaaatat
     6061 ttatttgtac ttaatttctt attttattaa ttaaatatgc tgatgtctaa acaaactatt
     6121 ttttttgttt ttggaaaact tatatttgag taatttagag aaagacaaat gctttttttt
     6181 ttgggttttt ttttttaaat gaaatttgtt ttacaaacga atagaaagta tagatgaagg
     6241 tgaaatacat atttgaattt tttttatata aaagattaaa gttgttggtt gttatgaaag
     6301 gttgaagtga agttggtttg aaggttgaag ttggatgaat gaatgaaagg ttgaagtttg
     6361 gtgaatgaat gaaaggttga agttggatga atgaaagttg aagttttggt ttgaatgaag
     6421 ggttttgtga aagtgatttg ttctttcttt acttgattat ttttggttat tgtttatgag
     6481 agtggattcg ctcttgcata tgataaaagg caaagaaaga aagatatttt taattaagga
     6541 aagcccgggg gatctgaatt cgatatcaag cttatcgata ccgtcgacct cgaggggggg
     6601 cccggtaccc aattcgccct atagtgagtc gtattacaat tcactggccg tcgttttaca
     6661 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     6721 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     6781 cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     6841 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     6901 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     6961 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     7021 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     7081 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     7141 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     7201 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     7261 g
//
