LOCUS       sequence-485667-        6553 bp    DNA     circular SYN 10-SEP-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 6553)
  TITLE     Tetracyclin Promoter swap system
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6553)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 10, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pFS554
FEATURES             Location/Qualifiers
     source          1..6553
                     /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="MSRLDKSKVINSALELLNEVGIEGLTTHKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     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(3585..4941)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(3585..3782)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(3788..4597)
                     /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     4511..4530
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4598..4941)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        complement(5054..5072)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5055..5072)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5153..5172)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5272..5294
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5332..5350)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5418..5522
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5523..5591,5592..6383)
                     /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(5741..5760)
                     /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 ataaactcgc 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 tatcgaatcg acagcagtat agcgaccagc
     3601 attcacatac gattgacgca tgatattact ttctgcgcac ttaacttcgc atctgggcag
     3661 atgatgtcga ggcgaaaaaa aatataaatc acgctaacat ttgattaaaa tagaacaact
     3721 acaatataaa aaaactatac aaatgacaag ttcttgaaaa caagaatctt tttattgtca
     3781 gtactgatta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     3841 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     3901 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     3961 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     4021 tgacgactga atccggtgag aatggcaaaa gcttatgcat ttctttccag acttgttcaa
     4081 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     4141 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     4201 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     4261 caggatattc ttctaatacc tggaatgctg ttttgccggg gatcgcagtg gtgagtaacc
     4321 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     4381 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     4441 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     4501 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     4561 atcgcggcct cgaaacgtga gtcttttcct tacccatggt tgtttatgtt cggatgtgat
     4621 gtgagaactg tatcctagca agattttaaa aggaagtata tgaaagaaga acctcagtgg
     4681 caaatcctaa ccttttatat ttctctacag gggcgcggcg tggggacaat tcaacgcgtc
     4741 tgtgagggga gcgtttccct gctcgcaggt ctgcagcgag gagccgtaat ttttgcttcg
     4801 cgccgtgcgg ccatcaaaat gtatggatgc aaatgattat acatggggat gtatgggcta
     4861 aatgtacggg cgacagtcac atcatgcccc tgagctgcgc acgtcaagac tgtcaaggag
     4921 ggtattctgg gcctccatgt cgctggccgg gtgacccggc ggggacgagg caagctaaac
     4981 agatctggcg cgccttaatt aacccgggga tccgtcgacc tgcagcgtac gaagcttcag
     5041 ctggcggccg cgttctatag tgtcacctaa atcgtatgtg tatgatacat aaggttatgt
     5101 attaattgta gccgcgttct aacgacaata tgtccatatg gtgcactctc agtacaatct
     5161 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     5221 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     5281 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     5341 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     5401 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5461 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5521 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     5581 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     5641 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     5701 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     5761 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     5821 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     5881 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     5941 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     6001 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     6061 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     6121 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     6181 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     6241 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     6301 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     6361 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6421 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6481 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6541 tcaaaggatc ttc
//
