LOCUS       sequence-384626-        7244 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tetracycline inducible expression of msfGFP fused to Tat signal 
            peptide in Staphylococci.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7244)
  AUTHORS   Evans DCS, Khamas AB, Marcussen L, Rasmussen KS, Klitgaard JK, 
            Kallipolitis BH, Nielsen J, Otzen DE, Leake MC, Meyer RL
  TITLE     GFP fusions of Sec-routed extracellular proteins in Staphylococcus 
            aureus reveal surface-associated coagulase in biofilms.
  JOURNAL   Microb Cell. 2023 Jun 28;10(7):145-156. doi: 
            10.15698/mic2023.07.800. eCollection 2023 Jul 3.
  PUBMED    37395997
REFERENCE   2  (bases 1 to 7244)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.15698/mic2023.07.800"; journalName: "Microb Cell"; date: 
            "2023-06-28- 28"; volume: "10"; issue: "7"; pages: "145-156"
COMMENT     Sequence Label: pRMC2-tat:msfGFP
FEATURES             Location/Qualifiers
     source          1..7244
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..40)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     140..159
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     353..375
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     367..384
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     368..384
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1251..1269
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     CDS             1376..2002
                     /codon_start=1
                     /gene="tetR"
                     /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="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(1452..1471)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             complement(2314..2328)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     primer_bind     complement(5012..5028)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5012..5028)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5025..5047)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5036..5052
                     /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)."
     promoter        complement(join(5060..5066,5067..5084,5085..5090))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5105..5126
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5243..5260)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5414..6002)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5494..5513)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6173..6964,6965..7033))
                     /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     6796..6815
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7034..7138)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7206..7224
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
       61 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
      121 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg
      181 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca ttcgccattc
      241 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      301 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      361 cgacgttgta aaacgacggc cagtgaattc ttatttatat aattcatcca taccatgtgt
      421 aatacctgct gctgtaacaa attctaataa aaccatatga tcacgttttt catttggatc
      481 ttttgataat tttgattgtg ttgataaata atgattatct ggtaataaaa ctggaccatc
      541 accaattggt gtattttgtt gataatgatc tgctaattga actgaaccat cttcaacatt
      601 atgacgaatt ttaaaatttg ctttaatacc atttttttgt ttatctgctg taatataaac
      661 attatgtgaa ttaaaattat attctaattt atgacctaaa atattaccat cttctttaaa
      721 atcaatacct tttaattcaa tacgattaac taatgtatca ccttcaaatt taacttctgc
      781 acgtgtttta tatgtaccat catctttaaa tgaaattgta cgttcttgaa cataaccttc
      841 tggcattgct gatttaaaaa aatcatgttg tttcatatga tctggataac gtgaaaaaca
      901 ttgaacacca tatgttaatg ttgtaactaa tgttggccat ggaactggta atttacctgt
      961 tgtacaaata aattttaatg ttaatttacc atttgttgca tcaccttcac cttcaccacg
     1021 aactgaaaat ttatgaccat taacatcacc atctaattca actaaaattg gaacaacacc
     1081 tgtaaataat tcttcacctt ttgatcctcc accacctgat gcaccaattg caacacctgc
     1141 accaccaata cctaacattt ttaaaaatgt acgacgtgaa aattgtgttg aatcattaac
     1201 ttgttcataa tttgtcatgg taccctcctc atcaagctta ttttaattat actctatcaa
     1261 tgatagagtg tcaatatttt ttttagtttt tcatgaactc gaggggatcc aaataaaaaa
     1321 ctagtttgac aaataactct atcaatgata taatgtcaac aaaaaggagg aattaatgat
     1381 gtctagatta gataaaagta aagtgattaa cagcgcatta gagctgctta atgaggtcgg
     1441 aatcgaaggt ttaacaaccc gtaaactcgc ccagaagcta ggtgtagagc agcctacatt
     1501 gtattggcat gtaaaaaata agcgggcttt gctcgacgcc ttagccattg agatgttaga
     1561 taggcaccat actcactttt gccctttaga aggggaaagc tggcaagatt ttttacgtaa
     1621 taacgctaaa agttttagat gtgctttact aagtcatcgc gatggagcaa aagtacattt
     1681 aggtacacgg cctacagaaa aacagtatga aactctcgaa aatcaattag cctttttatg
     1741 ccaacaaggt ttttcactag agaatgcatt atatgcactc agcgctgtgg ggcattttac
     1801 tttaggttgc gtattggaag atcaagagca tcaagtcgct aaagaagaaa gggaaacacc
     1861 tactactgat agtatgccgc cattattacg acaagctatc gaattatttg atcaccaagg
     1921 tgcagagcca gccttcttat tcggccttga attgatcata tgcggattag aaaaacaact
     1981 taaatgtgaa agtgggtctt aaaagcagca taaccttttt ccgtgatggt aacttcacgg
     2041 taaccaagat gtcgagttat cgataccgtc gacctgcagg catgcaagct tttaaaaagc
     2101 aaatatgagc caaataaata tattctaatt ctacaaacaa aaatttgagc aaattcagtg
     2161 tcgatttttt aagacactgc ccagttacat gcaaattaaa attttcatga ttttttatag
     2221 ttcctaacag ggttaaaatt tgtataacga aagtataatg tttatataac gttagtataa
     2281 taaagcattt taacattata cttttgataa tcgtttatcg tcgtcatcac aataactttt
     2341 aaaatactcg tgcataattc aacagctgac ctcccaataa ctacatggtg ttatcgggag
     2401 gtcagctgtt agcacttata ttttgttatt gttcttcctc gatttcgtct atcattttgt
     2461 gattaatttc tcttttttct tgttctgtta agtcataaag ttcactagct aaatactctt
     2521 tttgtttcca aatataaaaa atttgataga tatattcggt tggatcaatt tcttttaagt
     2581 aatctaaatc cccatttttt aatttctttt tagcctcttt aaataatcct gaataaacta
     2641 atacctgttt acctttaagt gatttataaa atgcatcaaa gactttttga tttattaaat
     2701 aatcactatc tttaccagaa tacttagcca tttcatataa ttctttatta ttattttgtc
     2761 ttattttttg aacttgaact tgtgttattt ctgaaatgcc cgttacatca cgccataaat
     2821 ctaaccattc ttgttggcta atataatatc ttttatctgt gaaatacgat ttatttactg
     2881 caattaacac atgaaaatga ggattataat catctctttt tttattatat gtaatctcta
     2941 acttacgaac atatcccttt ataacactac ctactttttt tctctttata agttttctaa
     3001 aagaattatt ataacgtttt atttcatttt ctaattcatc actcattaca ttaggtgtag
     3061 tcaaagttaa aaagataaac tcctttttct cttgctgctt aatatattgc atcatcaaag
     3121 ataaacccaa tgcatctttt ctagcttttc tccaagcaca gacaggacaa aatcgatttt
     3181 tacaagaatt agctttatat aatttctgtt tttctaaagt tttatcagct acaaaagaca
     3241 gaaatgtatt gcaatcttca actaaatcca tttgattctc tccaatatga cgtttaataa
     3301 atttctgaaa tacttgattt ctttgttttt tctcagtata cttttccatg ttataacaca
     3361 taaaaacaac ttagttttca caaactatga caataaaaaa agttgctttt tcccctttct
     3421 atgtatgttt tttactattc atttaaaacg atacattaat aggtacgaaa aagcaacttt
     3481 ttttgcgctt aaaaccagtc ataccaataa cttaagggta actagcctcg ccggcaatag
     3541 ttacccttat tatcaagata agaaagaaaa ggatttttcg ctacgctcaa atcctttaaa
     3601 aaaacacaaa agaccacatt ttttaatgtg gtcttttatt cttcaactaa agcacccatt
     3661 agttcaacaa acgaaaattg gataaagtgg gatattttta aaatatatat ttatgttaca
     3721 gtaatattga cttttaaaaa aggattgatt ctaatgaaga aagcagacaa gtaagcctcc
     3781 taaattcact ttagataaaa atttaggagg catatcaaat gaactttaat aaaattgatt
     3841 tagacaattg gaagagaaaa gagatattta atcattattt gaaccaacaa acgactttta
     3901 gtataaccac agaaattgat attagtgttt tataccgaaa cataaaacaa gaaggatata
     3961 aattttaccc tgcatttatt ttcttagtga caagggtgat aaactcaaat acagctttta
     4021 gaactggtta caatagcgac ggagagttag gttattggga taagttagag ccactttata
     4081 caatttttga tggtgtatct aaaacattct ctggtatttg gactcctgta aagaatgact
     4141 tcaaagagtt ttatgattta tacctttctg atgtagagaa atataatggt tcggggaaat
     4201 tgtttcccaa aacacctata cctgaaaatg ctttttctct ttctattatt ccatggactt
     4261 catttactgg gtttaactta aatatcaata ataatagtaa ttaccttcta cccattatta
     4321 cagcaggaaa attcattaat aaaggtaatt caatatattt accgctatct ttacaggtac
     4381 atcattctgt ttgtgatggt tatcatgcag gattgtttat gaactctatt caggaattgt
     4441 cagataggcc taatgactgg cttttataat atgagataat gccgactgta ctttttacag
     4501 tcggttttct aatgtcacta acctgccccg ttagttgaag aaggttttta tattacagct
     4561 ccagatccat atccttcttt ttctgaaccg acttctcctt tttcgcttct ttattccaat
     4621 tgctttattg acgttgagcc tcggaaccct taacaatccc aaaacttgtc gaatggtcgg
     4681 cttaatagct cacgctatgc cgacattcgt ctgcaagttt agttaagggt tcttctcaac
     4741 gcacaataaa ttttctcggc ataaatgcgt ggtctaattt ttatttttaa taaccttgat
     4801 agcaaaaaat gccattccaa tacaaaacca catacctata atcgataacc acataacagt
     4861 cataaaacca ctccttttta acaaacttta tcacaagaaa tatttaaatt ttaaatgcct
     4921 ttattttgaa ttttaagggg cattttaaag atttaggggt aaatcatata gttttatgcc
     4981 taaaaaccta cagaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     5041 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     5101 cctaatgagt gagctaactc acattaattg cgttgcgctc actgtccgct ttccagtcgg
     5161 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5221 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     5281 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5341 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5401 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5461 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5521 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5581 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5641 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5701 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5761 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5821 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5881 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5941 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     6001 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     6061 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6121 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6181 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6241 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6301 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6361 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6421 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6481 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6541 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6601 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6661 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6721 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6781 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6841 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6901 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6961 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     7021 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     7081 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     7141 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     7201 ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttc
//
