LOCUS       sequence-484485-        6555 bp    DNA     circular SYN 25-AUG-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 6555)
  TITLE     Tetracyclin Promoter swap system
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6555)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 25, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pFS555
FEATURES             Location/Qualifiers
     source          1..6555
                     /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    1730..1748
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     enhancer        2056..2435
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2436..2639
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2590..2610
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2636..2660
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2741..3364
                     /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(2814..2833)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(3374..3392)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3402..3419)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3402..3418)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3411..3433)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     gene            complement(3587..4943)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(3587..3784)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(3790..4599)
                     /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     4513..4532
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4600..4943)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        complement(5056..5074)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5057..5074)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5155..5174)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5274..5296
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5334..5352)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5420..5524
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5525..5593,5594..6385)
                     /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(5743..5762)
                     /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 gggcccgagc
     1681 ggcccgcgat gtttactgta gaatactaat tgattcttac tgaaacaagt ctctatcact
     1741 gatagggaca ctctatatat acctggagga agcgccgatc ctctcaatct tccatgacat
     1801 gcccctgagc ccacagaagc cggtggaaca aattggtcgc ttctttaaga atcgttaacc
     1861 ggaacgttag ggtacgttga aggagcgacc aagcgagcga cgcgagccac cgtagccaat
     1921 gtgcgagtcc agcgagctta gaaggggcaa gagatggggc ccggggggtc gaggagcttg
     1981 gcccattgca tacgttgtat ccatatcata atatgtacat ttatattggc tcatgtccaa
     2041 cattaccgcc atgttgacat tgattattga ctagttatta atagtaatca attacggggt
     2101 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     2161 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     2221 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     2281 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     2341 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     2401 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     2461 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     2521 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     2581 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc
     2641 gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa
     2701 gacaccggga ccgatccagc ctccgcggcc ccgaattcat atgtctagat tagataaaag
     2761 taaagtgatt aacagcgcat tagagctgct taatgaggtc ggaatcgaag gtttaacaac
     2821 ccgtaaactc gcccagaagc taggtgtaga gcagcctaca ttgtattggc atgtaaaaaa
     2881 taagcgggct ttgctcgacg ccttagccat tgagatgtta gataggcacc atactcactt
     2941 ttgcccttta gaaggggaaa gctggcaaga ttttttacgt aataacgcta aaagttttag
     3001 atgtgcttta ctaagtcatc gcgatggagc aaaagtacat ttaggtacac ggcctacaga
     3061 aaaacagtat gaaactctcg aaaatcaatt agccttttta tgccaacaag gtttttcact
     3121 agagaatgca ttatatgcac tcagcgctgt ggggcatttt actttaggtt gcgtattgga
     3181 agatcaagag catcaagtcg ctaaagaaga aagggaaaca cctactactg atagtatgcc
     3241 gccattatta cgacaagcta tcgaattatt tgatcaccaa ggtgcagagc cagccttctt
     3301 attcggcctt gaattgatca tatgcggatt agaaaaacaa cttaaatgtg aaagtggatc
     3361 ttaaccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
     3421 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     3481 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     3541 agcctgaatg gcgaatggga tggcggcgtt agtatcgaat cgacagcagt atagcgacca
     3601 gcattcacat acgattgacg catgatatta ctttctgcgc acttaacttc gcatctgggc
     3661 agatgatgtc gaggcgaaaa aaaatataaa tcacgctaac atttgattaa aatagaacaa
     3721 ctacaatata aaaaaactat acaaatgaca agttcttgaa aacaagaatc tttttattgt
     3781 cagtactgat tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     3841 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     3901 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     3961 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     4021 agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc agacttgttc
     4081 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     4141 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     4201 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     4261 atcaggatat tcttctaata cctggaatgc tgttttgccg gggatcgcag tggtgagtaa
     4321 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     4381 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     4441 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     4501 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     4561 taatcgcggc ctcgaaacgt gagtcttttc cttacccatg gttgtttatg ttcggatgtg
     4621 atgtgagaac tgtatcctag caagatttta aaaggaagta tatgaaagaa gaacctcagt
     4681 ggcaaatcct aaccttttat atttctctac aggggcgcgg cgtggggaca attcaacgcg
     4741 tctgtgaggg gagcgtttcc ctgctcgcag gtctgcagcg aggagccgta atttttgctt
     4801 cgcgccgtgc ggccatcaaa atgtatggat gcaaatgatt atacatgggg atgtatgggc
     4861 taaatgtacg ggcgacagtc acatcatgcc cctgagctgc gcacgtcaag actgtcaagg
     4921 agggtattct gggcctccat gtcgctggcc gggtgacccg gcggggacga ggcaagctaa
     4981 acagatctgg cgcgccttaa ttaacccggg gatccgtcga cctgcagcgt acgaagcttc
     5041 agctggcggc cgcgttctat agtgtcacct aaatcgtatg tgtatgatac ataaggttat
     5101 gtattaattg tagccgcgtt ctaacgacaa tatgtccata tggtgcactc tcagtacaat
     5161 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     5221 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     5281 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     5341 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     5401 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5461 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5521 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     5581 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     5641 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     5701 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     5761 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     5821 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     5881 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     5941 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     6001 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     6061 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     6121 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     6181 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     6241 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     6301 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     6361 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     6421 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     6481 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6541 gatcaaagga tcttc
//
