LOCUS       sequence-486279-        7774 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid will be used to replace the promoter of a gene with the
            PenotetS promoter at its endogenous locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7774)
  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 7774)
  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: "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: pFS561
FEATURES             Location/Qualifiers
     source          1..7774
                     /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     complement(1135..1155)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     1750..1778
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     protein_bind    1858..1876
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     enhancer        2184..2563
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2564..2767
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2718..2738
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2764..2788
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2869..3492
                     /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(2942..2961)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(3502..3520)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3530..3546)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     gene            complement(3715..4834)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(3715..3912)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(3918..4490)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(4491..4834)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        complement(4978..4996)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5003..5019)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      5160..5588
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5247..5266)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5457..5478
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             5932..6726
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5986..6005)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6596..6615
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(6744..6812,6813..7604)
                     /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(6962..6981)
                     /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 gccaagcttg gtaccgagct
     1021 cggatccact agtaacggcc gccagtgtgc tggaattcgc ccttgaattc gagctcgttt
     1081 aaactgtaag atcttttgta tagttcatcc atgccatgtg taatcccagc agctgttaca
     1141 aactcaagaa ggaccatgtg gtctctcttt tcgttgggat ctttcgaaag cttagattga
     1201 taggacaggt aatggttgtc tggtaaaagg acagggccat cgccaattgg agtattttgt
     1261 tgataatggt ctgctagttg aacgcttcca tcttcaatgt tgtgtctaat tttgaagtta
     1321 actttgattc cattcttttg tttgtctgcc atgatgtata cattgtgtga gttatagttg
     1381 tattccaatt tgtgtccaag aatgtttcca tcttctttaa aatcaatacc ttttaactcg
     1441 attctattaa caagggtatc accttcaaac ttgacttcag cacgtgtctt gtagttcccg
     1501 tcatctttga aaaatatagt tctttcctgt acataacctt cgggcatggc actcttgaaa
     1561 aagtcatgcc gtttcatatg atctgggtat ctagcaaagc acattagacc ataacccaaa
     1621 gtagtgacaa gtgttggcca tggaacaggt agttttccag tagtgcaaat aaatttaagg
     1681 gtaagttttc cgtatgttgc atcaccttca ccctctccac tgacagaaaa tttgtgccca
     1741 ttaacatcac catctaattc aacaagaatt gggacaactc cagtgaaaag ttcttctcct
     1801 ttactcatgc tagccgatgt ttactgtaga atactaattg attcttactg aaacaagtct
     1861 ctatcactga tagggacact ctatatatac ctggaggaag cgccgatcct ctcaatcttc
     1921 catgacatgc ccctgagccc acagaagccg gtggaacaaa ttggtcgctt ctttaagaat
     1981 cgttaaccgg aacgttaggg tacgttgaag gagcgaccaa gcgagcgacg cgagccaccg
     2041 tagccaatgt gcgagtccag cgagcttaga aggggcaaga gatggggccc ggggggtcga
     2101 ggagcttggc ccattgcata cgttgtatcc atatcataat atgtacattt atattggctc
     2161 atgtccaaca ttaccgccat gttgacattg attattgact agttattaat agtaatcaat
     2221 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
     2281 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     2341 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     2401 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     2461 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     2521 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
     2581 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
     2641 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
     2701 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
     2761 cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct
     2821 ccatagaaga caccgggacc gatccagcct ccgcggcccc gaattcatat gtctagatta
     2881 gataaaagta aagtgattaa cagcgcatta gagctgctta atgaggtcgg aatcgaaggt
     2941 ttaacaaccc gtaaactcgc ccagaagcta ggtgtagagc agcctacatt gtattggcat
     3001 gtaaaaaata agcgggcttt gctcgacgcc ttagccattg agatgttaga taggcaccat
     3061 actcactttt gccctttaga aggggaaagc tggcaagatt ttttacgtaa taacgctaaa
     3121 agttttagat gtgctttact aagtcatcgc gatggagcaa aagtacattt aggtacacgg
     3181 cctacagaaa aacagtatga aactctcgaa aatcaattag cctttttatg ccaacaaggt
     3241 ttttcactag agaatgcatt atatgcactc agcgctgtgg ggcattttac tttaggttgc
     3301 gtattggaag atcaagagca tcaagtcgct aaagaagaaa gggaaacacc tactactgat
     3361 agtatgccgc cattattacg acaagctatc gaattatttg atcaccaagg tgcagagcca
     3421 gccttcttat tcggccttga attgatcata tgcggattag aaaaacaact taaatgtgaa
     3481 agtggatctt aaccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg
     3541 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     3601 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     3661 agttgcgcag cctgaatggc gaatgggatg gcggcgttag tatcgaatcg acagcagtat
     3721 agcgaccagc attcacatac gattgacgca tgatattact ttctgcgcac ttaacttcgc
     3781 atctgggcag atgatgtcga ggcgaaaaaa aatataaatc acgctaacat ttgattaaaa
     3841 tagaacaact acaatataaa aaaactatac aaatgacaag ttcttgaaaa caagaatctt
     3901 tttattgtca gtactgatta ggggcagggc atgctcatgt agagcgcctg ctcgccgtcc
     3961 gaggcggtgc cgtcgtacag ggcggtgtcc aggccgcaga gggtgaaccc catccgccgg
     4021 tacgcgtgga tcgccggtgc gttgacgttg gtgacctcca gccagaggtg cccggcgccc
     4081 cgctcgcggg cgaactccgt cgcgagcccc atcaacgcgc gcccgacccc gtgcccccgg
     4141 tgctccgggg cgacctcgat gtcctcgacg gtcagccggc ggttccagcc ggagtacgag
     4201 acgaccacga agcccgccag gtcgccgtcg tccccgtacg cgacgaacgt ccgggagtcc
     4261 gggtcgccgt cctccccgtc gtccgattcg tcgtccgatt cgtcgtcggg gaacaccttg
     4321 gtcaggggcg ggtccaccgg cacctcccgc agggtgaagc cgtccccggt ggcggtgacg
     4381 cggaagacgg tgtcggtggt gaaggaccca tccagtgcct cgatggcctc ggcgtccccc
     4441 gggacactgg tgcggtaccg gtaagccgtg tcgtcaagag tggtacccat ggttgtttat
     4501 gttcggatgt gatgtgagaa ctgtatccta gcaagatttt aaaaggaagt atatgaaaga
     4561 agaacctcag tggcaaatcc taacctttta tatttctcta caggggcgcg gcgtggggac
     4621 aattcaacgc gtctgtgagg ggagcgtttc cctgctcgca ggtctgcagc gaggagccgt
     4681 aatttttgct tcgcgccgtg cggccatcaa aatgtatgga tgcaaatgat tatacatggg
     4741 gatgtgtggg ctaaatatac gggcgacagt cacatcatgc ccctgagctg cgcacgtcaa
     4801 gactgtcaag gagggtattc tgggcctcca tgtcgctggc cgggtgaccc ggcggggacg
     4861 aggcaagcta aacagatctg gcgcgcctta attaacccgg ggatccgaag ggcgaattct
     4921 gcagatatcc atcacactgg cggccgctcg agcatgcatc tagagggccc aattcgccct
     4981 atagtgagtc gtattacaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     5041 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     5101 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
     5161 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     5221 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     5281 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     5341 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     5401 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     5461 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     5521 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     5581 cgcgaatttt aacaaaattc agggcgcaag ggctgctaaa ggaagcggaa cacgtagaaa
     5641 gccagtccgc agaaacggtg ctgaccccgg atgaatgtca gctactgggc tatctggaca
     5701 agggaaaacg caagcgcaaa gagaaagcag gtagcttgca gtgggcttac atggcgatag
     5761 ctagactggg cggttttatg gacagcaagc gaaccggaat tgccagctgg ggcgccctct
     5821 ggtaaggttg ggaagccctg caaagtaaac tggatggctt tcttgccgcc aaggatctga
     5881 tggcgcaggg gatcaagatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     5941 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     6001 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     6061 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     6121 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     6181 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     6241 tcatcccacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     6301 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     6361 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     6421 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     6481 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     6541 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     6601 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     6661 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     6721 ttctgaattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     6781 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     6841 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     6901 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     6961 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     7021 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     7081 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     7141 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     7201 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     7261 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     7321 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     7381 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     7441 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     7501 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     7561 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     7621 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     7681 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     7741 cgtcagaccc cgtagaaaag atcaaaggat cttc
//
