LOCUS       sequence-485676-        7908 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid will be used to replace the promoter of a gene with the
            PenotetSW1 promoter at its endogenous locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7908)
  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 7908)
  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: pFS562
FEATURES             Location/Qualifiers
     source          1..7908
                     /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    1869..1887
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     enhancer        2195..2574
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2575..2778
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2729..2749
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2775..2799
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2880..3503
                     /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(2953..2972)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(3513..3531)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3541..3557)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     gene            complement(3849..4968)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(3849..4046)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(4052..4624)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(4625..4968)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        complement(5112..5130)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5137..5153)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      5294..5722
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5381..5400)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5591..5612
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             6066..6860
                     /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(6120..6139)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6730..6749
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(6878..6946,6947..7738)
                     /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(7096..7115)
                     /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 tagcgcccga gcggccgatg tttactgtag aatactaatt gattcttact
     1861 gaaacaagtc tctatcactg atagggacac tctatatata cctggaggaa gcgccgatcc
     1921 tctcaatctt ccatgacatg cccctgagcc cacagaagcc ggtggaacaa attggtcgct
     1981 tctttaagaa tcgttaaccg gaacgttagg gtacgttgaa ggagcgacca agcgagcgac
     2041 gcgagccacc gtagccaatg tgcgagtcca gcgagcttag aaggggcaag agatggggcc
     2101 cggggggtcg aggagcttgg cccattgcat acgttgtatc catatcataa tatgtacatt
     2161 tatattggct catgtccaac attaccgcca tgttgacatt gattattgac tagttattaa
     2221 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     2281 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     2341 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     2401 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     2461 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     2521 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
     2581 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
     2641 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
     2701 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
     2761 gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc
     2821 tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccc cgaattcata
     2881 tgtctagatt agataaaagt aaagtgatta acagcgcatt agagctgctt aatgaggtcg
     2941 gaatcgaagg tttaacaacc cgtaaactcg cccagaagct aggtgtagag cagcctacat
     3001 tgtattggca tgtaaaaaat aagcgggctt tgctcgacgc cttagccatt gagatgttag
     3061 ataggcacca tactcacttt tgccctttag aaggggaaag ctggcaagat tttttacgta
     3121 ataacgctaa aagttttaga tgtgctttac taagtcatcg cgatggagca aaagtacatt
     3181 taggtacacg gcctacagaa aaacagtatg aaactctcga aaatcaatta gcctttttat
     3241 gccaacaagg tttttcacta gagaatgcat tatatgcact cagcgctgtg gggcatttta
     3301 ctttaggttg cgtattggaa gatcaagagc atcaagtcgc taaagaagaa agggaaacac
     3361 ctactactga tagtatgccg ccattattac gacaagctat cgaattattt gatcaccaag
     3421 gtgcagagcc agccttctta ttcggccttg aattgatcat atgcggatta gaaaaacaac
     3481 ttaaatgtga aagtggatct taaccaattc gccctatagt gagtcgtatt acgcgcgctc
     3541 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     3601 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     3661 cccttcccaa cagttgcgca gcctgaatgg cgaatgggat ggcggcgtta gtatctccac
     3721 tagtaacggc cgccagtgtg ctggaattcg cccttgaatt cgagctcgtt taaactgtaa
     3781 gatcttttgt atagttcatc catgcctgaa tggcgaatgg gatggcggcg ttagtatcga
     3841 atcgacagca gtatagcgac cagcattcac atacgattga cgcatgatat tactttctgc
     3901 gcacttaact tcgcatctgg gcagatgatg tcgaggcgaa aaaaaatata aatcacgcta
     3961 acatttgatt aaaatagaac aactacaata taaaaaaact atacaaatga caagttcttg
     4021 aaaacaagaa tctttttatt gtcagtactg attaggggca gggcatgctc atgtagagcg
     4081 cctgctcgcc gtccgaggcg gtgccgtcgt acagggcggt gtccaggccg cagagggtga
     4141 accccatccg ccggtacgcg tggatcgccg gtgcgttgac gttggtgacc tccagccaga
     4201 ggtgcccggc gccccgctcg cgggcgaact ccgtcgcgag ccccatcaac gcgcgcccga
     4261 ccccgtgccc ccggtgctcc ggggcgacct cgatgtcctc gacggtcagc cggcggttcc
     4321 agccggagta cgagacgacc acgaagcccg ccaggtcgcc gtcgtccccg tacgcgacga
     4381 acgtccggga gtccgggtcg ccgtcctccc cgtcgtccga ttcgtcgtcc gattcgtcgt
     4441 cggggaacac cttggtcagg ggcgggtcca ccggcacctc ccgcagggtg aagccgtccc
     4501 cggtggcggt gacgcggaag acggtgtcgg tggtgaagga cccatccagt gcctcgatgg
     4561 cctcggcgtc ccccgggaca ctggtgcggt accggtaagc cgtgtcgtca agagtggtac
     4621 ccatggttgt ttatgttcgg atgtgatgtg agaactgtat cctagcaaga ttttaaaagg
     4681 aagtatatga aagaagaacc tcagtggcaa atcctaacct tttatatttc tctacagggg
     4741 cgcggcgtgg ggacaattca acgcgtctgt gaggggagcg tttccctgct cgcaggtctg
     4801 cagcgaggag ccgtaatttt tgcttcgcgc cgtgcggcca tcaaaatgta tggatgcaaa
     4861 tgattataca tggggatgtg tgggctaaat atacgggcga cagtcacatc atgcccctga
     4921 gctgcgcacg tcaagactgt caaggagggt attctgggcc tccatgtcgc tggccgggtg
     4981 acccggcggg gacgaggcaa gctaaacaga tctggcgcgc cttaattaac ccggggatcc
     5041 gaagggcgaa ttctgcagat atccatcaca ctggcggccg ctcgagcatg catctagagg
     5101 gcccaattcg ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg
     5161 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     5221 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     5281 gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     5341 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     5401 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     5461 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     5521 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     5581 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     5641 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     5701 taacaaaaat ttaacgcgaa ttttaacaaa attcagggcg caagggctgc taaaggaagc
     5761 ggaacacgta gaaagccagt ccgcagaaac ggtgctgacc ccggatgaat gtcagctact
     5821 gggctatctg gacaagggaa aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc
     5881 ttacatggcg atagctagac tgggcggttt tatggacagc aagcgaaccg gaattgccag
     5941 ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt aaactggatg gctttcttgc
     6001 cgccaaggat ctgatggcgc aggggatcaa gatctgatca agagacagga tgaggatcgt
     6061 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     6121 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     6181 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     6241 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     6301 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     6361 ggcaggatct cctgtcatcc caccttgctc ctgccgagaa agtatccatc atggctgatg
     6421 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     6481 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     6541 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     6601 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     6661 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     6721 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     6781 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     6841 ttcttgacga gttcttctga attgaaaaag gaagagtatg agtattcaac atttccgtgt
     6901 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     6961 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     7021 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     7081 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     7141 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     7201 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     7261 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     7321 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     7381 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     7441 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     7501 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     7561 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     7621 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     7681 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     7741 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     7801 aaggatctag gtgaagatcc ttttngataa tctcatgacc aaaatccctt aacgtgagtt
     7861 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttc
//
