LOCUS       sequence-478730-        6538 bp    DNA     circular SYN 15-JUL-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 6538)
  TITLE     Tetracyclin Promoter swap system
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6538)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pFS551
FEATURES             Location/Qualifiers
     source          1..6538
                     /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    1713..1731
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     enhancer        2039..2418
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2419..2622
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2573..2593
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2619..2643
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2724..3347
                     /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(2797..2816)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(3357..3375)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3385..3402)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3385..3401)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3394..3416)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     gene            complement(3570..4926)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(3570..3767)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(3773..4582)
                     /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     4496..4515
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4583..4926)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        complement(5039..5057)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5040..5057)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5138..5157)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5257..5279
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5317..5335)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5403..5507
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5508..5576,5577..6368)
                     /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(5726..5745)
                     /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 catgctagcc gatgtttact
     1681 gtagaatact aattgattct tactgaaaca agtctctatc actgataggg acactctata
     1741 tatacctgga ggaagcgccg atcctctcaa tcttccatga catgcccctg agcccacaga
     1801 agccggtgga acaaattggt cgcttcttta agaatcgtta accggaacgt tagggtacgt
     1861 tgaaggagcg accaagcgag cgacgcgagc caccgtagcc aatgtgcgag tccagcgagc
     1921 ttagaagggg caagagatgg ggcccggggg gtcgaggagc ttggcccatt gcatacgttg
     1981 tatccatatc ataatatgta catttatatt ggctcatgtc caacattacc gccatgttga
     2041 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
     2101 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
     2161 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
     2221 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     2281 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     2341 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     2401 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
     2461 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
     2521 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
     2581 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctcgtttagt gaaccgtcag
     2641 atcgcctgga gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc
     2701 agcctccgcg gccccgaatt catatgtcta gattagataa aagtaaagtg attaacagcg
     2761 cattagagct gcttaatgag gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga
     2821 agctaggtgt agagcagcct acattgtatt ggcatgtaaa aaataagcgg gctttgctcg
     2881 acgccttagc cattgagatg ttagataggc accatactca cttttgccct ttagaagggg
     2941 aaagctggca agatttttta cgtaataacg ctaaaagttt tagatgtgct ttactaagtc
     3001 atcgcgatgg agcaaaagta catttaggta cacggcctac agaaaaacag tatgaaactc
     3061 tcgaaaatca attagccttt ttatgccaac aaggtttttc actagagaat gcattatatg
     3121 cactcagcgc tgtggggcat tttactttag gttgcgtatt ggaagatcaa gagcatcaag
     3181 tcgctaaaga agaaagggaa acacctacta ctgatagtat gccgccatta ttacgacaag
     3241 ctatcgaatt atttgatcac caaggtgcag agccagcctt cttattcggc cttgaattga
     3301 tcatatgcgg attagaaaaa caacttaaat gtgaaagtgg atcttaacca attcgcccta
     3361 tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa
     3421 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     3481 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     3541 ggatggcggc gttagtatcg aatcgacagc agtatagcga ccagcattca catacgattg
     3601 acgcatgata ttactttctg cgcacttaac ttcgcatctg ggcagatgat gtcgaggcga
     3661 aaaaaaatat aaatcacgct aacatttgat taaaatagaa caactacaat ataaaaaaac
     3721 tatacaaatg acaagttctt gaaaacaaga atctttttat tgtcagtact gattagaaaa
     3781 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     3841 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     3901 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     3961 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     4021 gtgagaatgg caaaagctta tgcatttctt tccagacttg ttcaacaggc cagccattac
     4081 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     4141 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     4201 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     4261 atacctggaa tgctgttttg ccggggatcg cagtggtgag taaccatgca tcatcaggag
     4321 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     4381 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     4441 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     4501 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctcgaaa
     4561 cgtgagtctt ttccttaccc atggttgttt atgttcggat gtgatgtgag aactgtatcc
     4621 tagcaagatt ttaaaaggaa gtatatgaaa gaagaacctc agtggcaaat cctaaccttt
     4681 tatatttctc tacaggggcg cggcgtgggg acaattcaac gcgtctgtga ggggagcgtt
     4741 tccctgctcg caggtctgca gcgaggagcc gtaatttttg cttcgcgccg tgcggccatc
     4801 aaaatgtatg gatgcaaatg attatacatg gggatgtatg ggctaaatgt acgggcgaca
     4861 gtcacatcat gcccctgagc tgcgcacgtc aagactgtca aggagggtat tctgggcctc
     4921 catgtcgctg gccgggtgac ccggcgggga cgaggcaagc taaacagatc tggcgcgcct
     4981 taattaaccc ggggatccgt cgacctgcag cgtacgaagc ttcagctggc ggccgcgttc
     5041 tatagtgtca cctaaatcgt atgtgtatga tacataaggt tatgtattaa ttgtagccgc
     5101 gttctaacga caatatgtcc atatggtgca ctctcagtac aatctgctct gatgccgcat
     5161 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     5221 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     5281 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     5341 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     5401 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5461 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     5521 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5581 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5641 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5701 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     5761 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5821 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5881 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     5941 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     6001 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     6061 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     6121 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     6181 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     6241 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     6301 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6361 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     6421 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     6481 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttc
//
