LOCUS       sequence-484488-        6551 bp    DNA     circular SYN 25-AUG-2025
DEFINITION  This plasmid will be used to replace the promoter of a gene with the
            PenotetSW2 promoter at its endogenous locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6551)
  TITLE     Tetracyclin Promoter swap system
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6551)
  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            "Unpublished"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: pFS553
FEATURES             Location/Qualifiers
     source          1..6551
                     /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    1726..1744
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     enhancer        2052..2431
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2432..2635
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2586..2606
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2632..2656
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2737..3360
                     /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(2810..2829)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(3370..3388)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3398..3415)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3398..3414)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3407..3429)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     gene            complement(3583..4939)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(3583..3780)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(3786..4595)
                     /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     4509..4528
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4596..4939)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        complement(5052..5070)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5053..5070)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5151..5170)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5270..5292
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5330..5348)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5416..5520
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5521..5589,5590..6381)
                     /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(5739..5758)
                     /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 gcccgagcgg
     1681 cccgatgttt actgtagaat actaattgat tcttactgaa acaagtctct atcactgata
     1741 gggacactct atatatacct ggaggaagcg ccgatcctct caatcttcca tgacatgccc
     1801 ctgagcccac agaagccggt ggaacaaatt ggtcgcttct ttaagaatcg ttaaccggaa
     1861 cgttagggta cgttgaagga gcgaccaagc gagcgacgcg agccaccgta gccaatgtgc
     1921 gagtccagcg agcttagaag gggcaagaga tggggcccgg ggggtcgagg agcttggccc
     1981 attgcatacg ttgtatccat atcataatat gtacatttat attggctcat gtccaacatt
     2041 accgccatgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
     2101 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     2161 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     2221 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     2281 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     2341 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     2401 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
     2461 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     2521 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     2581 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt
     2641 agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca
     2701 ccgggaccga tccagcctcc gcggccccga attcatatgt ctagattaga taaaagtaaa
     2761 gtgattaaca gcgcattaga gctgcttaat gaggtcggaa tcgaaggttt aacaacccgt
     2821 aaactcgccc agaagctagg tgtagagcag cctacattgt attggcatgt aaaaaataag
     2881 cgggctttgc tcgacgcctt agccattgag atgttagata ggcaccatac tcacttttgc
     2941 cctttagaag gggaaagctg gcaagatttt ttacgtaata acgctaaaag ttttagatgt
     3001 gctttactaa gtcatcgcga tggagcaaaa gtacatttag gtacacggcc tacagaaaaa
     3061 cagtatgaaa ctctcgaaaa tcaattagcc tttttatgcc aacaaggttt ttcactagag
     3121 aatgcattat atgcactcag cgctgtgggg cattttactt taggttgcgt attggaagat
     3181 caagagcatc aagtcgctaa agaagaaagg gaaacaccta ctactgatag tatgccgcca
     3241 ttattacgac aagctatcga attatttgat caccaaggtg cagagccagc cttcttattc
     3301 ggccttgaat tgatcatatg cggattagaa aaacaactta aatgtgaaag tggatcttaa
     3361 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc
     3421 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     3481 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     3541 tgaatggcgg atgggatggc ggcgttagta tcgaatcgac agcagtatag cgaccagcat
     3601 tcacatacga ttgacgcatg atattacttt ctgcgcactt aacttcgcat ctgggcagat
     3661 gatgtcgagg cgaaaaaaaa tataaatcac gctaacattt gattaaaata gaacaactac
     3721 aatataaaaa aactatacaa atgacaagtt cttgaaaaca agaatctttt tattgtcagt
     3781 actgattaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     3841 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     3901 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     3961 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     4021 acgactgaat ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca
     4081 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     4141 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     4201 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     4261 ggatattctt ctaatacctg gaatgctgtt ttgccgggga tcgcagtggt gagtaaccat
     4321 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     4381 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     4441 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     4501 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     4561 cgcggcctcg aaacgtgagt cttttcctta cccatggttg tttatgttcg gatgtgatgt
     4621 gagaactgta tcctagcaag attttaaaag gaagtatatg aaagaagaac ctcagtggca
     4681 aatcctaacc ttttatattt ctctacaggg gcgcggcgtg gggacaattc aacgcgtctg
     4741 tgaggggagc gtttccctgc tcgcaggtct gcagcgagga gccgtaattt ttgcttcgcg
     4801 ccgtgcggcc atcaaaatgt atggatgcaa atgattatac atggggatgt atgggctaaa
     4861 tgtacgggcg acagtcacat catgcccctg agctgcgcac gtcaagactg tcaaggaggg
     4921 tattctgggc ctccatgtcg ctggccgggt gacccggcgg ggacgaggca agctaaacag
     4981 atctggcgcg ccttaattaa cccggggatc cgtcgacctg cagcgtacga agcttcagct
     5041 ggcggccgcg ttctatagtg tcacctaaat cgtatgtgta tgatacataa ggttatgtat
     5101 taattgtagc cgcgttctaa cgacaatatg tccatatggt gcactctcag tacaatctgc
     5161 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     5221 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     5281 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     5341 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     5401 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5461 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5521 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5581 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5641 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5701 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5761 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5821 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5881 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5941 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     6001 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     6061 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     6121 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     6181 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     6241 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6301 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6361 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6421 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     6481 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6541 aaaggatctt c
//
