LOCUS       sequence-384624-        7133 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tetracycline inducible expression of msfGFP in Staphylococci.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7133)
  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 7133)
  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-msfGFP
FEATURES             Location/Qualifiers
     source          1..7133
                     /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    1140..1158
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     CDS             1265..1891
                     /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(1341..1360)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             complement(2203..2217)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     primer_bind     complement(4901..4917)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4901..4917)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4914..4936)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4925..4941
                     /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(4949..4955,4956..4973,4974..4979))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4994..5015
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5132..5149)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5303..5891)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5383..5402)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6062..6853,6854..6922))
                     /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     6685..6704
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6923..7027)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7095..7113
                     /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 ttgacatggt accctcctca tcaagcttat tttaattata
     1141 ctctatcaat gatagagtgt caatattttt tttagttttt catgaactcg aggggatcca
     1201 aataaaaaac tagtttgaca aataactcta tcaatgatat aatgtcaaca aaaaggagga
     1261 attaatgatg tctagattag ataaaagtaa agtgattaac agcgcattag agctgcttaa
     1321 tgaggtcgga atcgaaggtt taacaacccg taaactcgcc cagaagctag gtgtagagca
     1381 gcctacattg tattggcatg taaaaaataa gcgggctttg ctcgacgcct tagccattga
     1441 gatgttagat aggcaccata ctcacttttg ccctttagaa ggggaaagct ggcaagattt
     1501 tttacgtaat aacgctaaaa gttttagatg tgctttacta agtcatcgcg atggagcaaa
     1561 agtacattta ggtacacggc ctacagaaaa acagtatgaa actctcgaaa atcaattagc
     1621 ctttttatgc caacaaggtt tttcactaga gaatgcatta tatgcactca gcgctgtggg
     1681 gcattttact ttaggttgcg tattggaaga tcaagagcat caagtcgcta aagaagaaag
     1741 ggaaacacct actactgata gtatgccgcc attattacga caagctatcg aattatttga
     1801 tcaccaaggt gcagagccag ccttcttatt cggccttgaa ttgatcatat gcggattaga
     1861 aaaacaactt aaatgtgaaa gtgggtctta aaagcagcat aacctttttc cgtgatggta
     1921 acttcacggt aaccaagatg tcgagttatc gataccgtcg acctgcaggc atgcaagctt
     1981 ttaaaaagca aatatgagcc aaataaatat attctaattc tacaaacaaa aatttgagca
     2041 aattcagtgt cgatttttta agacactgcc cagttacatg caaattaaaa ttttcatgat
     2101 tttttatagt tcctaacagg gttaaaattt gtataacgaa agtataatgt ttatataacg
     2161 ttagtataat aaagcatttt aacattatac ttttgataat cgtttatcgt cgtcatcaca
     2221 ataactttta aaatactcgt gcataattca acagctgacc tcccaataac tacatggtgt
     2281 tatcgggagg tcagctgtta gcacttatat tttgttattg ttcttcctcg atttcgtcta
     2341 tcattttgtg attaatttct cttttttctt gttctgttaa gtcataaagt tcactagcta
     2401 aatactcttt ttgtttccaa atataaaaaa tttgatagat atattcggtt ggatcaattt
     2461 cttttaagta atctaaatcc ccatttttta atttcttttt agcctcttta aataatcctg
     2521 aataaactaa tacctgttta cctttaagtg atttataaaa tgcatcaaag actttttgat
     2581 ttattaaata atcactatct ttaccagaat acttagccat ttcatataat tctttattat
     2641 tattttgtct tattttttga acttgaactt gtgttatttc tgaaatgccc gttacatcac
     2701 gccataaatc taaccattct tgttggctaa tataatatct tttatctgtg aaatacgatt
     2761 tatttactgc aattaacaca tgaaaatgag gattataatc atctcttttt ttattatatg
     2821 taatctctaa cttacgaaca tatcccttta taacactacc tacttttttt ctctttataa
     2881 gttttctaaa agaattatta taacgtttta tttcattttc taattcatca ctcattacat
     2941 taggtgtagt caaagttaaa aagataaact cctttttctc ttgctgctta atatattgca
     3001 tcatcaaaga taaacccaat gcatcttttc tagcttttct ccaagcacag acaggacaaa
     3061 atcgattttt acaagaatta gctttatata atttctgttt ttctaaagtt ttatcagcta
     3121 caaaagacag aaatgtattg caatcttcaa ctaaatccat ttgattctct ccaatatgac
     3181 gtttaataaa tttctgaaat acttgatttc tttgtttttt ctcagtatac ttttccatgt
     3241 tataacacat aaaaacaact tagttttcac aaactatgac aataaaaaaa gttgcttttt
     3301 cccctttcta tgtatgtttt ttactattca tttaaaacga tacattaata ggtacgaaaa
     3361 agcaactttt tttgcgctta aaaccagtca taccaataac ttaagggtaa ctagcctcgc
     3421 cggcaatagt tacccttatt atcaagataa gaaagaaaag gatttttcgc tacgctcaaa
     3481 tcctttaaaa aaacacaaaa gaccacattt tttaatgtgg tcttttattc ttcaactaaa
     3541 gcacccatta gttcaacaaa cgaaaattgg ataaagtggg atatttttaa aatatatatt
     3601 tatgttacag taatattgac ttttaaaaaa ggattgattc taatgaagaa agcagacaag
     3661 taagcctcct aaattcactt tagataaaaa tttaggaggc atatcaaatg aactttaata
     3721 aaattgattt agacaattgg aagagaaaag agatatttaa tcattatttg aaccaacaaa
     3781 cgacttttag tataaccaca gaaattgata ttagtgtttt ataccgaaac ataaaacaag
     3841 aaggatataa attttaccct gcatttattt tcttagtgac aagggtgata aactcaaata
     3901 cagcttttag aactggttac aatagcgacg gagagttagg ttattgggat aagttagagc
     3961 cactttatac aatttttgat ggtgtatcta aaacattctc tggtatttgg actcctgtaa
     4021 agaatgactt caaagagttt tatgatttat acctttctga tgtagagaaa tataatggtt
     4081 cggggaaatt gtttcccaaa acacctatac ctgaaaatgc tttttctctt tctattattc
     4141 catggacttc atttactggg tttaacttaa atatcaataa taatagtaat taccttctac
     4201 ccattattac agcaggaaaa ttcattaata aaggtaattc aatatattta ccgctatctt
     4261 tacaggtaca tcattctgtt tgtgatggtt atcatgcagg attgtttatg aactctattc
     4321 aggaattgtc agataggcct aatgactggc ttttataata tgagataatg ccgactgtac
     4381 tttttacagt cggttttcta atgtcactaa cctgccccgt tagttgaaga aggtttttat
     4441 attacagctc cagatccata tccttctttt tctgaaccga cttctccttt ttcgcttctt
     4501 tattccaatt gctttattga cgttgagcct cggaaccctt aacaatccca aaacttgtcg
     4561 aatggtcggc ttaatagctc acgctatgcc gacattcgtc tgcaagttta gttaagggtt
     4621 cttctcaacg cacaataaat tttctcggca taaatgcgtg gtctaatttt tatttttaat
     4681 aaccttgata gcaaaaaatg ccattccaat acaaaaccac atacctataa tcgataacca
     4741 cataacagtc ataaaaccac tcctttttaa caaactttat cacaagaaat atttaaattt
     4801 taaatgcctt tattttgaat tttaaggggc attttaaaga tttaggggta aatcatatag
     4861 ttttatgcct aaaaacctac agaagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     4921 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4981 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgtccgctt
     5041 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     5101 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     5161 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5221 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5281 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5341 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5401 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5461 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5521 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5581 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5641 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5701 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5761 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5821 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5881 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5941 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     6001 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6061 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6121 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6181 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6241 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6301 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6361 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6421 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6481 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6541 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6601 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6661 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6721 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6781 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6841 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6901 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6961 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     7021 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     7081 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttc
//
