LOCUS       sequence-486278-        8230 bp    DNA     circular SYN 16-SEP-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 8230)
  TITLE     Tetracyclin Promoter swap system
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8230)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pFS556
FEATURES             Location/Qualifiers
     source          1..8230
                     /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"
     gene            1138..2713
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        1138..1481
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1482..2510
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLMHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      2516..2713
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     2882..2898
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2908..2926
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(2936..3559)
                     /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     3467..3486
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3640..3664)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(3661..3864)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3690..3710)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3865..4244
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    4552..4570
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(4650..4678)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     5273..5293
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     promoter        complement(5434..5452)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5459..5475)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      5616..6044
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5703..5722)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5913..5934
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             6388..7182
                     /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(6442..6461)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7052..7071
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(7200..7268,7269..8060)
                     /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(7418..7437)
                     /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 ccttcggatc cccgggttaa
     1081 ttaaggcgcg ccagatctgt ttagcttgcc tcgtccccgc cgggtcaccc ggccagcgac
     1141 atggaggccc agaataccct ccttgacagt cttgacgtgc gcagctcagg ggcatgatgt
     1201 gactgtcgcc cgtacattta gcccatacat ccccatgtat aatcatttgc atccatacat
     1261 tttgatggcc gcacggcgcg aagcaaaaat tacggctcct cgctgcggac ctgcgagcag
     1321 ggaaacgctc ccctcacaga cgcgttgaat tgtccccacg ccgcgcccct gtagagaaat
     1381 ataaaaggtt aggatttgcc actgaggttc ttctttcata tacttccttt taaaatcttg
     1441 ctaggataca gttctcacat cacatccgaa cataaacaac catgggtaaa aagcctgaac
     1501 tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga
     1561 tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat
     1621 atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc
     1681 actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga
     1741 gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa
     1801 ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg
     1861 atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta
     1921 catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga
     1981 tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg
     2041 aggactgccc cgaagtccgg cacctcatgc acgcggattt cggctccaac aatgtcctga
     2101 cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc
     2161 aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga
     2221 cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata
     2281 tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg
     2341 cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc
     2401 gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg
     2461 ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaataa tcagtactga
     2521 caataaaaag attcttgttt tcaagaactt gtcatttgta tagttttttt atattgtagt
     2581 tgttctattt taatcaaatg ttagcgtgat ttatattttt tttcgcctcg acatcatctg
     2641 cccagatgcg aagttaagtg cgcagaaagt aatatcatgc gtcaatcgta tgtgaatgct
     2701 ggtcgctata ctgctgtcga ttcgatacta acgccgccat cccattcgcc attcaggctg
     2761 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
     2821 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt
     2881 tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac tatagggcga attggttaag
     2941 atccactttc acatttaagt tgtttttcta atccgcatat gatcaattca aggccgaata
     3001 agaaggctgg ctctgcacct tggtgatcaa ataattcgat agcttgtcgt aataatggcg
     3061 gcatactatc agtagtaggt gtttcccttt cttctttagc gacttgatgc tcttgatctt
     3121 ccaatacgca acctaaagta aaatgcccca cagcgctgag tgcatataat gcattctcta
     3181 gtgaaaaacc ttgttggcat aaaaaggcta attgattttc gagagtttca tactgttttt
     3241 ctgtaggccg tgtacctaaa tgtacttttg ctccatcgcg atgacttagt aaagcacatc
     3301 taaaactttt agcgttatta cgtaaaaaat cttgccagct ttccccttct aaagggcaaa
     3361 agtgagtatg gtgcctatct aacatctcaa tggctaaggc gtcgagcaaa gcccgcttat
     3421 tttttacatg ccaatacaat gtaggctgct ctacacctag cttctgggcg agtttacggg
     3481 ttgttaaacc ttcgattccg acctcattaa gcagctctaa tgcgctgtta atcactttac
     3541 ttttatctaa tctagacata tgaattcggg gccgcggagg ctggatcggt cccggtgtct
     3601 tctatggagg tcaaaacagc gtggatggcg tctccaggcg atctgacggt tcactaaacg
     3661 agctctgctt atatagacct cccaccgtac acgcctaccg cccatttgcg tcaatggggc
     3721 ggagttgtta cgacattttg gaaagtcccg ttgattttgg tgccaaaaca aactcccatt
     3781 gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt
     3841 gatgtactgc caaaaccgca tcaccatggt aatagcgatg actaatacgt agatgtactg
     3901 ccaagtagga aagtcccata aggtcatgta ctgggcataa tgccaggcgg gccatttacc
     3961 gtcattgacg tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg
     4021 ggcagtttac cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac
     4081 tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg
     4141 cgggccattt accgtaagtt atgtaacgcg gaactccata tatgggctat gaactaatga
     4201 ccccgtaatt gattactatt aataactagt caataatcaa tgtcaacatg gcggtaatgt
     4261 tggacatgag ccaatataaa tgtacatatt atgatatgga tacaacgtat gcaatgggcc
     4321 aagctcctcg accccccggg ccccatctct tgccccttct aagctcgctg gactcgcaca
     4381 ttggctacgg tggctcgcgt cgctcgcttg gtcgctcctt caacgtaccc taacgttccg
     4441 gttaacgatt cttaaagaag cgaccaattt gttccaccgg cttctgtggg ctcaggggca
     4501 tgtcatggaa gattgagagg atcggcgctt cctccaggta tatatagagt gtccctatca
     4561 gtgatagaga cttgtttcag taagaatcaa ttagtattct acagtaaaca tcggctagca
     4621 tgagtaaagg agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg
     4681 atgttaatgg gcacaaattt tctgtcagtg gagagggtga aggtgatgca acatacggaa
     4741 aacttaccct taaatttatt tgcactactg gaaaactacc tgttccatgg ccaacacttg
     4801 tcactacttt gggttatggt ctaatgtgct ttgctagata cccagatcat atgaaacggc
     4861 atgacttttt caagagtgcc atgcccgaag gttatgtaca ggaaagaact atatttttca
     4921 aagatgacgg gaactacaag acacgtgctg aagtcaagtt tgaaggtgat acccttgtta
     4981 atagaatcga gttaaaaggt attgatttta aagaagatgg aaacattctt ggacacaaat
     5041 tggaatacaa ctataactca cacaatgtat acatcatggc agacaaacaa aagaatggaa
     5101 tcaaagttaa cttcaaaatt agacacaaca ttgaagatgg aagcgttcaa ctagcagacc
     5161 attatcaaca aaatactcca attggcgatg gccctgtcct tttaccagac aaccattacc
     5221 tgtcctatca atctaagctt tcgaaagatc ccaacgaaaa gagagaccac atggtccttc
     5281 ttgagtttgt aacagctgct gggattacac atggcatgga tgaactatac aaaagatctt
     5341 acagtttaaa cgagctcgaa ttcaagggcg aattctgcag atatccatca cactggcggc
     5401 cgctcgagca tgcatctaga gggcccaatt cgccctatag tgagtcgtat tacaattcac
     5461 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     5521 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     5581 cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag
     5641 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     5701 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     5761 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     5821 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     5881 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     5941 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     6001 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaattcaggg
     6061 cgcaagggct gctaaaggaa gcggaacacg tagaaagcca gtccgcagaa acggtgctga
     6121 ccccggatga atgtcagcta ctgggctatc tggacaaggg aaaacgcaag cgcaaagaga
     6181 aagcaggtag cttgcagtgg gcttacatgg cgatagctag actgggcggt tttatggaca
     6241 gcaagcgaac cggaattgcc agctggggcg ccctctggta aggttgggaa gccctgcaaa
     6301 gtaaactgga tggctttctt gccgccaagg atctgatggc gcaggggatc aagatctgat
     6361 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     6421 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     6481 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     6541 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     6601 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     6661 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat cccaccttgc tcctgccgag
     6721 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     6781 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     6841 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     6901 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     6961 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     7021 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     7081 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     7141 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gaattgaaaa aggaagagta
     7201 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     7261 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     7321 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     7381 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     7441 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     7501 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     7561 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     7621 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     7681 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     7741 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     7801 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     7861 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     7921 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     7981 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     8041 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     8101 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     8161 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     8221 aaggatcttc
//
