LOCUS       sequence-491391-        6645 bp    DNA     circular SYN 03-NOV-2025
DEFINITION  This plasmid will be used to replace the promoter of a gene with the
            PenotetSW3 promoter at its endogenous locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6645)
  AUTHORS   Bashir S, Sivakumar S, Rhind N
  TITLE     A Tetracycline-Inducible Promoter Replacement System for 
            Schizosaccharomyces pombe.
  JOURNAL   MicroPubl Biol. 2025 Aug 21;2025:10.17912/micropub.biology.001759. 
            doi: 10.17912/micropub.biology.001759. eCollection 2025.
  PUBMED    40918427
REFERENCE   2  (bases 1 to 6645)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 3, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "MicroPubl 
            Biol."; date: "2025-08-21"; volume: 
            "2025:10.17912/micropub.biology.001759. doi"; pages: " 
            10.17912/micropub.biology.001759. eCollection 2025"
COMMENT     Sequence Label: pFS554
FEATURES             Location/Qualifiers
     source          1..6645
                     /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"
     promoter        847..865
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(990..1010)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     1605..1633
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     protein_bind    1728..1746
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     enhancer        2054..2433
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2434..2637
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2588..2608
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2634..2658
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2739..3362
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(2812..2831)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(3372..3390)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3400..3417)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3400..3416)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3409..3431)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     gene            complement(3677..5033)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(3677..3874)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(3880..4689)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4603..4622
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4690..5033)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        complement(5146..5164)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5147..5164)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5245..5264)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5364..5386
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5424..5442)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5510..5614
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5615..5683,5684..6475)
                     /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(5833..5852)
                     /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 tgcaggttaa cctggcttat
      841 cgaaattaat acgactcact atagggagac cggcagatcc gcggccgcat aggccactag
      901 tggatctgat atcatcgatg aattcgagct cgtttaaact gtaagatctt ttgtatagtt
      961 catccatgcc atgtgtaatc ccagcagctg ttacaaactc aagaaggacc atgtggtctc
     1021 tcttttcgtt gggatctttc gaaagcttag attgatagga caggtaatgg ttgtctggta
     1081 aaaggacagg gccatcgcca attggagtat tttgttgata atggtctgct agttgaacgc
     1141 ttccatcttc aatgttgtgt ctaattttga agttaacttt gattccattc ttttgtttgt
     1201 ctgccatgat gtatacattg tgtgagttat agttgtattc caatttgtgt ccaagaatgt
     1261 ttccatcttc tttaaaatca atacctttta actcgattct attaacaagg gtatcacctt
     1321 caaacttgac ttcagcacgt gtcttgtagt tcccgtcatc tttgaaaaat atagttcttt
     1381 cctgtacata accttcgggc atggcactct tgaaaaagtc atgccgtttc atatgatctg
     1441 ggtatctagc aaagcacatt agaccataac ccaaagtagt gacaagtgtt ggccatggaa
     1501 caggtagttt tccagtagtg caaataaatt taagggtaag ttttccgtat gttgcatcac
     1561 cttcaccctc tccactgaca gaaaatttgt gcccattaac atcaccatct aattcaacaa
     1621 gaattgggac aactccagtg aaaagttctt ctcctttact catgctagcg ggcccgagcg
     1681 gccccgatgt ttactgtaga atactaattg attcttactg aaacaagtct ctatcactga
     1741 tagggacact ctatatatac ctggaggaag cgccgatcct ctcaatcttc catgacatgc
     1801 ccctgagccc acagaagccg gtggaacaaa ttggtcgctt ctttaagaat cgttaaccgg
     1861 aacgttaggg tacgttgaag gagcgaccaa gcgagcgacg cgagccaccg tagccaatgt
     1921 gcgagtccag cgagcttaga aggggcaaga gatggggccc ggggggtcga ggagcttggc
     1981 ccattgcata cgttgtatcc atatcataat atgtacattt atattggctc atgtccaaca
     2041 ttaccgccat gttgacattg attattgact agttattaat agtaatcaat tacggggtca
     2101 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     2161 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     2221 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     2281 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     2341 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     2401 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
     2461 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     2521 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     2581 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
     2641 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
     2701 caccgggacc gatccagcct ccgcggcccc gaattcatat gtctagatta gataaaagta
     2761 aagtgattaa cagcgcatta gagctgctta atgaggtcgg aatcgaaggt ttaacaaccc
     2821 gtaaactcgc ccagaagcta ggtgtagagc agcctacatt gtattggcat gtaaaaaata
     2881 agcgggcttt gctcgacgcc ttagccattg agatgttaga taggcaccat actcactttt
     2941 gccctttaga aggggaaagc tggcaagatt ttttacgtaa taacgctaaa agttttagat
     3001 gtgctttact aagtcatcgc gatggagcaa aagtacattt aggtacacgg cctacagaaa
     3061 aacagtatga aactctcgaa aatcaattag cctttttatg ccaacaaggt ttttcactag
     3121 agaatgcatt atatgcactc agcgctgtgg ggcattttac tttaggttgc gtattggaag
     3181 atcaagagca tcaagtcgct aaagaagaaa gggaaacacc tactactgat agtatgccgc
     3241 cattattacg acaagctatc gaattatttg atcaccaagg tgcagagcca gccttcttat
     3301 tcggccttga attgatcata tgcggattag aaaaacaact taaatgtgaa agtggatctt
     3361 aaccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg
     3421 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     3481 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     3541 cctgaatggc gaatgggatg gcggcgttag tataacagac catggaattc gagctcgttt
     3601 aaactgtaag atcttttgta tagttcatca tgcctgaatg gcgaatggga tggcggcgtt
     3661 agtatcgaat cgacagcagt atagcgacca gcattcacat acgattgacg catgatatta
     3721 ctttctgcgc acttaacttc gcatctgggc agatgatgtc gaggcgaaaa aaaatataaa
     3781 tcacgctaac atttgattaa aatagaacaa ctacaatata aaaaaactat acaaatgaca
     3841 agttcttgaa aacaagaatc tttttattgt cagtactgat tagaaaaact catcgagcat
     3901 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     3961 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     4021 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     4081 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     4141 aagcttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     4201 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     4261 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     4321 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     4381 tgttttgccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     4441 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     4501 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     4561 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     4621 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctcgaaacgt gagtcttttc
     4681 cttacccatg gttgtttatg ttcggatgtg atgtgagaac tgtatcctag caagatttta
     4741 aaaggaagta tatgaaagaa gaacctcagt ggcaaatcct aaccttttat atttctctac
     4801 aggggcgcgg cgtggggaca attcaacgcg tctgtgaggg gagcgtttcc ctgctcgcag
     4861 gtctgcagcg aggagccgta atttttgctt cgcgccgtgc ggccatcaaa atgtatggat
     4921 gcaaatgatt atacatgggg atgtatgggc taaatgtacg ggcgacagtc acatcatgcc
     4981 cctgagctgc gcacgtcaag actgtcaagg agggtattct gggcctccat gtcgctggcc
     5041 gggtgacccg gcggggacga ggcaagctaa acagatctgg cgcgccttaa ttaacccggg
     5101 gatccgtcga cctgcagcgt acgaagcttc agctggcggc cgcgttctat agtgtcacct
     5161 aaatcgtatg tgtatgatac ataaggttat gtattaattg tagccgcgtt ctaacgacaa
     5221 tatgtccata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     5281 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     5341 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     5401 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     5461 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     5521 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5581 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     5641 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5701 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5761 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     5821 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     5881 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     5941 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     6001 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     6061 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     6121 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     6181 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     6241 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     6301 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     6361 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     6421 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     6481 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     6541 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6601 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttc
//
