LOCUS       sequence-485663-        8243 bp    DNA     circular SYN 10-SEP-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 8243)
  TITLE     Tetracyclin Promoter swap system
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8243)
  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            "Unpublished"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: pFS558
FEATURES             Location/Qualifiers
     source          1..8243
                     /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(4663..4691)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     5286..5306
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     promoter        complement(5447..5465)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5472..5488)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      5629..6057
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5716..5735)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5926..5947
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             6401..7195
                     /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(6455..6474)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7065..7084
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(7213..7281,7282..8073)
                     /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(7431..7450)
                     /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 tcgggccgct
     4621 cgggccgcta gcatgagtaa aggagaagaa cttttcactg gagttgtccc aattcttgtt
     4681 gaattagatg gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat
     4741 gcaacatacg gaaaacttac ccttaaattt atttgcacta ctggaaaact acctgttcca
     4801 tggccaacac ttgtcactac tttgggttat ggtctaatgt gctttgctag atacccagat
     4861 catatgaaac ggcatgactt tttcaagagt gccatgcccg aaggttatgt acaggaaaga
     4921 actatatttt tcaaagatga cgggaactac aagacacgtg ctgaagtcaa gtttgaaggt
     4981 gatacccttg ttaatagaat cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt
     5041 cttggacaca aattggaata caactataac tcacacaatg tatacatcat ggcagacaaa
     5101 caaaagaatg gaatcaaagt taacttcaaa attagacaca acattgaaga tggaagcgtt
     5161 caactagcag accattatca acaaaatact ccaattggcg atggccctgt ccttttacca
     5221 gacaaccatt acctgtccta tcaatctaag ctttcgaaag atcccaacga aaagagagac
     5281 cacatggtcc ttcttgagtt tgtaacagct gctgggatta cacatggcat ggatgaacta
     5341 tacaaaagat cttacagttt aaacgagctc gaattcaagg gcgaattctg cagatatcca
     5401 tcacactggc ggccgctcga gcatgcatct agagggccca attcgcccta tagtgagtcg
     5461 tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     5521 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     5581 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggac gcgccctgta
     5641 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     5701 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     5761 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     5821 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     5881 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     5941 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     6001 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     6061 acaaaattca gggcgcaagg gctgctaaag gaagcggaac acgtagaaag ccagtccgca
     6121 gaaacggtgc tgaccccgga tgaatgtcag ctactgggct atctggacaa gggaaaacgc
     6181 aagcgcaaag agaaagcagg tagcttgcag tgggcttaca tggcgatagc tagactgggc
     6241 ggttttatgg acagcaagcg aaccggaatt gccagctggg gcgccctctg gtaaggttgg
     6301 gaagccctgc aaagtaaact ggatggcttt cttgccgcca aggatctgat ggcgcagggg
     6361 atcaagatct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     6421 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     6481 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     6541 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
     6601 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     6661 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catcccacct
     6721 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     6781 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     6841 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     6901 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     6961 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     7021 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     7081 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     7141 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgaattga
     7201 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     7261 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     7321 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     7381 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     7441 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     7501 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     7561 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     7621 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     7681 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     7741 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     7801 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     7861 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     7921 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     7981 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     8041 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     8101 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     8161 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     8221 gtagaaaaga tcaaaggatc ttc
//
