LOCUS       sequence-485651-        7905 bp    DNA     circular SYN 15-DEC-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 7905)
  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 7905)
  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: pFS564
FEATURES             Location/Qualifiers
     source          1..7905
                     /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    1873..1891
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     enhancer        2199..2578
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2579..2782
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2733..2753
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2779..2803
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2884..3507
                     /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(2957..2976)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(3517..3535)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3545..3561)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     gene            complement(3846..4965)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(3846..4043)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(4049..4621)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(4622..4965)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        complement(5109..5127)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5134..5150)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      5291..5719
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5378..5397)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5588..5609
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             6063..6857
                     /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(6117..6136)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6727..6746
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(6875..6943,6944..7735)
                     /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(7093..7112)
                     /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 tagcgggccc gagcggcccc gatgtttact gtagaatact aattgattct
     1861 tactgaaaca agtctctatc actgataggg acactctata tatacctgga ggaagcgccg
     1921 atcctctcaa tcttccatga catgcccctg agcccacaga agccggtgga acaaattggt
     1981 cgcttcttta agaatcgtta accggaacgt tagggtacgt tgaaggagcg accaagcgag
     2041 cgacgcgagc caccgtagcc aatgtgcgag tccagcgagc ttagaagggg caagagatgg
     2101 ggcccggggg gtcgaggagc ttggcccatt gcatacgttg tatccatatc ataatatgta
     2161 catttatatt ggctcatgtc caacattacc gccatgttga cattgattat tgactagtta
     2221 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
     2281 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     2341 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     2401 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     2461 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
     2521 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     2581 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
     2641 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
     2701 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
     2761 ggaggtctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc
     2821 acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgcg gccccgaatt
     2881 catatgtcta gattagataa aagtaaagtg attaacagcg cattagagct gcttaatgag
     2941 gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt agagcagcct
     3001 acattgtatt ggcatgtaaa aaataagcgg gctttgctcg acgccttagc cattgagatg
     3061 ttagataggc accatactca cttttgccct ttagaagggg aaagctggca agatttttta
     3121 cgtaataacg ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg agcaaaagta
     3181 catttaggta cacggcctac agaaaaacag tatgaaactc tcgaaaatca attagccttt
     3241 ttatgccaac aaggtttttc actagagaat gcattatatg cactcagcgc tgtggggcat
     3301 tttactttag gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga agaaagggaa
     3361 acacctacta ctgatagtat gccgccatta ttacgacaag ctatcgaatt atttgatcac
     3421 caaggtgcag agccagcctt cttattcggc cttgaattga tcatatgcgg attagaaaaa
     3481 caacttaaat gtgaaagtgg atcttaacca attcgcccta tagtgagtcg tattacgcgc
     3541 gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     3601 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     3661 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggatggcggc gttagtatcg
     3721 aatcgacagc agtatagcga ccagcatatc gatgaattcg agctcgttta aactgtaaga
     3781 tcttttgtat agttcatcca gcctgaatgg cgaatgggat ggcggcgtta gtatcgaatc
     3841 gacagcagta tagcgaccag cattcacata cgattgacgc atgatattac tttctgcgca
     3901 cttaacttcg catctgggca gatgatgtcg aggcgaaaaa aaatataaat cacgctaaca
     3961 tttgattaaa atagaacaac tacaatataa aaaaactata caaatgacaa gttcttgaaa
     4021 acaagaatct ttttattgtc agtactgatt aggggcaggg catgctcatg tagagcgcct
     4081 gctcgccgtc cgaggcggtg ccgtcgtaca gggcggtgtc caggccgcag agggtgaacc
     4141 ccatccgccg gtacgcgtgg atcgccggtg cgttgacgtt ggtgacctcc agccagaggt
     4201 gcccggcgcc ccgctcgcgg gcgaactccg tcgcgagccc catcaacgcg cgcccgaccc
     4261 cgtgcccccg gtgctccggg gcgacctcga tgtcctcgac ggtcagccgg cggttccagc
     4321 cggagtacga gacgaccacg aagcccgcca ggtcgccgtc gtccccgtac gcgacgaacg
     4381 tccgggagtc cgggtcgccg tcctccccgt cgtccgattc gtcgtccgat tcgtcgtcgg
     4441 ggaacacctt ggtcaggggc gggtccaccg gcacctcccg cagggtgaag ccgtccccgg
     4501 tggcggtgac gcggaagacg gtgtcggtgg tgaaggaccc atccagtgcc tcgatggcct
     4561 cggcgtcccc cgggacactg gtgcggtacc ggtaagccgt gtcgtcaaga gtggtaccca
     4621 tggttgttta tgttcggatg tgatgtgaga actgtatcct agcaagattt taaaaggaag
     4681 tatatgaaag aagaacctca gtggcaaatc ctaacctttt atatttctct acaggggcgc
     4741 ggcgtgggga caattcaacg cgtctgtgag gggagcgttt ccctgctcgc aggtctgcag
     4801 cgaggagccg taatttttgc ttcgcgccgt gcggccatca aaatgtatgg atgcaaatga
     4861 ttatacatgg ggatgtgtgg gctaaatata cgggcgacag tcacatcatg cccctgagct
     4921 gcgcacgtca agactgtcaa ggagggtatt ctgggcctcc atgtcgctgg ccgggtgacc
     4981 cggcggggac gaggcaagct aaacagatct ggcgcgcctt aattaacccg gggatccgaa
     5041 gggcgaattc tgcagatatc catcacactg gcggccgctc gagcatgcat ctagagggcc
     5101 caattcgccc tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga
     5161 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     5221 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     5281 tggcgaatgg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     5341 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     5401 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     5461 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     5521 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     5581 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     5641 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     5701 caaaaattta acgcgaattt taacaaaatt cagggcgcaa gggctgctaa aggaagcgga
     5761 acacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc agctactggg
     5821 ctatctggac aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc agtgggctta
     5881 catggcgata gctagactgg gcggttttat ggacagcaag cgaaccggaa ttgccagctg
     5941 gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa ctggatggct ttcttgccgc
     6001 caaggatctg atggcgcagg ggatcaagat ctgatcaaga gacaggatga ggatcgtttc
     6061 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     6121 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     6181 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     6241 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     6301 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     6361 aggatctcct gtcatcccac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     6421 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     6481 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     6541 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     6601 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     6661 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     6721 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     6781 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     6841 ttgacgagtt cttctgaatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     6901 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     6961 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     7021 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     7081 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     7141 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     7201 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     7261 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     7321 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     7381 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     7441 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     7501 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     7561 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     7621 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     7681 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     7741 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     7801 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     7861 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttc
//
