LOCUS       sequence-484489-        7903 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid will be used to replace the promoter of a gene with the
            PenotetSW4 promoter at its endogenous locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7903)
  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 7903)
  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: pFS565
FEATURES             Location/Qualifiers
     source          1..7903
                     /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(1247..1267)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     1862..1890
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     protein_bind    1987..2005
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     enhancer        2313..2692
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2693..2896
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2847..2867
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2893..2917
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2998..3621
                     /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(3071..3090)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(3631..3649)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3659..3675)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     gene            complement(3844..4963)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(3844..4041)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(4047..4619)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(4620..4963)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        complement(5107..5125)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5132..5148)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      5289..5717
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5376..5395)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5586..5607
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             6061..6855
                     /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(6115..6134)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6725..6744
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(6873..6941,6942..7733)
                     /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(7091..7110)
                     /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 tgaatggcga atgggatggc ggcgttagta
     1141 tcgaatcgac agcagtatag cgaccagcat atcgatgaat tcgagctcgt ttaaactgta
     1201 agatcttttg tatagttcat ccatgccatg tgtaatccca gcagctgtta caaactcaag
     1261 aaggaccatg tggtctctct tttcgttggg atctttcgaa agcttagatt gataggacag
     1321 gtaatggttg tctggtaaaa ggacagggcc atcgccaatt ggagtatttt gttgataatg
     1381 gtctgctagt tgaacgcttc catcttcaat gttgtgtcta attttgaagt taactttgat
     1441 tccattcttt tgtttgtctg ccatgatgta tacattgtgt gagttatagt tgtattccaa
     1501 tttgtgtcca agaatgtttc catcttcttt aaaatcaata ccttttaact cgattctatt
     1561 aacaagggta tcaccttcaa acttgacttc agcacgtgtc ttgtagttcc cgtcatcttt
     1621 gaaaaatata gttctttcct gtacataacc ttcgggcatg gcactcttga aaaagtcatg
     1681 ccgtttcata tgatctgggt atctagcaaa gcacattaga ccataaccca aagtagtgac
     1741 aagtgttggc catggaacag gtagttttcc agtagtgcaa ataaatttaa gggtaagttt
     1801 tccgtatgtt gcatcacctt caccctctcc actgacagaa aatttgtgcc cattaacatc
     1861 accatctaat tcaacaagaa ttgggacaac tccagtgaaa agttcttctc ctttactcat
     1921 gctagccggg cccgagcggc ccgcgatgtt tactgtagaa tactaattga ttcttactga
     1981 aacaagtctc tatcactgat agggacactc tatatatacc tggaggaagc gccgatcctc
     2041 tcaatcttcc atgacatgcc cctgagccca cagaagccgg tggaacaaat tggtcgcttc
     2101 tttaagaatc gttaaccgga acgttagggt acgttgaagg agcgaccaag cgagcgacgc
     2161 gagccaccgt agccaatgtg cgagtccagc gagcttagaa ggggcaagag atggggcccg
     2221 gggggtcgag gagcttggcc cattgcatac gttgtatcca tatcataata tgtacattta
     2281 tattggctca tgtccaacat taccgccatg ttgacattga ttattgacta gttattaata
     2341 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
     2401 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
     2461 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     2521 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
     2581 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
     2641 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
     2701 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     2761 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     2821 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
     2881 ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc atccacgctg
     2941 ttttgacctc catagaagac accgggaccg atccagcctc cgcggccccg aattcatatg
     3001 tctagattag ataaaagtaa agtgattaac agcgcattag agctgcttaa tgaggtcgga
     3061 atcgaaggtt taacaacccg taaactcgcc cagaagctag gtgtagagca gcctacattg
     3121 tattggcatg taaaaaataa gcgggctttg ctcgacgcct tagccattga gatgttagat
     3181 aggcaccata ctcacttttg ccctttagaa ggggaaagct ggcaagattt tttacgtaat
     3241 aacgctaaaa gttttagatg tgctttacta agtcatcgcg atggagcaaa agtacattta
     3301 ggtacacggc ctacagaaaa acagtatgaa actctcgaaa atcaattagc ctttttatgc
     3361 caacaaggtt tttcactaga gaatgcatta tatgcactca gcgctgtggg gcattttact
     3421 ttaggttgcg tattggaaga tcaagagcat caagtcgcta aagaagaaag ggaaacacct
     3481 actactgata gtatgccgcc attattacga caagctatcg aattatttga tcaccaaggt
     3541 gcagagccag ccttcttatt cggccttgaa ttgatcatat gcggattaga aaaacaactt
     3601 aaatgtgaaa gtggatctta accaattcgc cctatagtga gtcgtattac gcgcgctcac
     3661 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     3721 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     3781 cttcccaaca gttgcgcagc ctgaatggcg aatgggatgg cggcgttagt atcgaatcga
     3841 cagcagtata gcgaccagca ttcacatacg attgacgcat gatattactt tctgcgcact
     3901 taacttcgca tctgggcaga tgatgtcgag gcgaaaaaaa atataaatca cgctaacatt
     3961 tgattaaaat agaacaacta caatataaaa aaactataca aatgacaagt tcttgaaaac
     4021 aagaatcttt ttattgtcag tactgattag gggcagggca tgctcatgta gagcgcctgc
     4081 tcgccgtccg aggcggtgcc gtcgtacagg gcggtgtcca ggccgcagag ggtgaacccc
     4141 atccgccggt acgcgtggat cgccggtgcg ttgacgttgg tgacctccag ccagaggtgc
     4201 ccggcgcccc gctcgcgggc gaactccgtc gcgagcccca tcaacgcgcg cccgaccccg
     4261 tgcccccggt gctccggggc gacctcgatg tcctcgacgg tcagccggcg gttccagccg
     4321 gagtacgaga cgaccacgaa gcccgccagg tcgccgtcgt ccccgtacgc gacgaacgtc
     4381 cgggagtccg ggtcgccgtc ctccccgtcg tccgattcgt cgtccgattc gtcgtcgggg
     4441 aacaccttgg tcaggggcgg gtccaccggc acctcccgca gggtgaagcc gtccccggtg
     4501 gcggtgacgc ggaagacggt gtcggtggtg aaggacccat ccagtgcctc gatggcctcg
     4561 gcgtcccccg ggacactggt gcggtaccgg taagccgtgt cgtcaagagt ggtacccatg
     4621 gttgtttatg ttcggatgtg atgtgagaac tgtatcctag caagatttta aaaggaagta
     4681 tatgaaagaa gaacctcagt ggcaaatcct aaccttttat atttctctac aggggcgcgg
     4741 cgtggggaca attcaacgcg tctgtgaggg gagcgtttcc ctgctcgcag gtctgcagcg
     4801 aggagccgta atttttgctt cgcgccgtgc ggccatcaaa atgtatggat gcaaatgatt
     4861 atacatgggg atgtgtgggc taaatatacg ggcgacagtc acatcatgcc cctgagctgc
     4921 gcacgtcaag actgtcaagg agggtattct gggcctccat gtcgctggcc gggtgacccg
     4981 gcggggacga ggcaagctaa acagatctgg cgcgccttaa ttaacccggg gatccgaagg
     5041 gcgaattctg cagatatcca tcacactggc ggccgctcga gcatgcatct agagggccca
     5101 attcgcccta tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact
     5161 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     5221 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     5281 gcgaatggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     5341 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     5401 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     5461 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     5521 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     5581 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     5641 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     5701 aaaatttaac gcgaatttta acaaaattca gggcgcaagg gctgctaaag gaagcggaac
     5761 acgtagaaag ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag ctactgggct
     5821 atctggacaa gggaaaacgc aagcgcaaag agaaagcagg tagcttgcag tgggcttaca
     5881 tggcgatagc tagactgggc ggttttatgg acagcaagcg aaccggaatt gccagctggg
     5941 gcgccctctg gtaaggttgg gaagccctgc aaagtaaact ggatggcttt cttgccgcca
     6001 aggatctgat ggcgcagggg atcaagatct gatcaagaga caggatgagg atcgtttcgc
     6061 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     6121 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     6181 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     6241 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     6301 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     6361 gatctcctgt catcccacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     6421 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     6481 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     6541 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     6601 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     6661 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     6721 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     6781 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     6841 gacgagttct tctgaattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     6901 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     6961 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     7021 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     7081 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     7141 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     7201 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     7261 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     7321 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     7381 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     7441 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     7501 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     7561 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     7621 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     7681 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     7741 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     7801 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     7861 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttc
//
