LOCUS       sequence-384625-        7223 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Tetracycline inducible expression of msfGFP fused to Sec signal 
            peptide in Staphylococci.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7223)
  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 7223)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-sec:msfGFP
FEATURES             Location/Qualifiers
     source          1..7223
                     /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    1230..1248
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     CDS             1355..1981
                     /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(1431..1450)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             complement(2293..2307)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     primer_bind     complement(4991..5007)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4991..5007)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5004..5026)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5015..5031
                     /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(5039..5045,5046..5063,5064..5069))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5084..5105
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5222..5239)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5393..5981)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5473..5492)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6152..6943,6944..7012))
                     /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     6775..6794
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7013..7117)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7185..7203
                     /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 gcatgtgctg aatgatacca
     1141 acctgacata acaactaaaa taattgataa aaataatgtt ttaatacatt ttttcatggt
     1201 accctcctca tcaagcttat tttaattata ctctatcaat gatagagtgt caatattttt
     1261 tttagttttt catgaactcg aggggatcca aataaaaaac tagtttgaca aataactcta
     1321 tcaatgatat aatgtcaaca aaaaggagga attaatgatg tctagattag ataaaagtaa
     1381 agtgattaac agcgcattag agctgcttaa tgaggtcgga atcgaaggtt taacaacccg
     1441 taaactcgcc cagaagctag gtgtagagca gcctacattg tattggcatg taaaaaataa
     1501 gcgggctttg ctcgacgcct tagccattga gatgttagat aggcaccata ctcacttttg
     1561 ccctttagaa ggggaaagct ggcaagattt tttacgtaat aacgctaaaa gttttagatg
     1621 tgctttacta agtcatcgcg atggagcaaa agtacattta ggtacacggc ctacagaaaa
     1681 acagtatgaa actctcgaaa atcaattagc ctttttatgc caacaaggtt tttcactaga
     1741 gaatgcatta tatgcactca gcgctgtggg gcattttact ttaggttgcg tattggaaga
     1801 tcaagagcat caagtcgcta aagaagaaag ggaaacacct actactgata gtatgccgcc
     1861 attattacga caagctatcg aattatttga tcaccaaggt gcagagccag ccttcttatt
     1921 cggccttgaa ttgatcatat gcggattaga aaaacaactt aaatgtgaaa gtgggtctta
     1981 aaagcagcat aacctttttc cgtgatggta acttcacggt aaccaagatg tcgagttatc
     2041 gataccgtcg acctgcaggc atgcaagctt ttaaaaagca aatatgagcc aaataaatat
     2101 attctaattc tacaaacaaa aatttgagca aattcagtgt cgatttttta agacactgcc
     2161 cagttacatg caaattaaaa ttttcatgat tttttatagt tcctaacagg gttaaaattt
     2221 gtataacgaa agtataatgt ttatataacg ttagtataat aaagcatttt aacattatac
     2281 ttttgataat cgtttatcgt cgtcatcaca ataactttta aaatactcgt gcataattca
     2341 acagctgacc tcccaataac tacatggtgt tatcgggagg tcagctgtta gcacttatat
     2401 tttgttattg ttcttcctcg atttcgtcta tcattttgtg attaatttct cttttttctt
     2461 gttctgttaa gtcataaagt tcactagcta aatactcttt ttgtttccaa atataaaaaa
     2521 tttgatagat atattcggtt ggatcaattt cttttaagta atctaaatcc ccatttttta
     2581 atttcttttt agcctcttta aataatcctg aataaactaa tacctgttta cctttaagtg
     2641 atttataaaa tgcatcaaag actttttgat ttattaaata atcactatct ttaccagaat
     2701 acttagccat ttcatataat tctttattat tattttgtct tattttttga acttgaactt
     2761 gtgttatttc tgaaatgccc gttacatcac gccataaatc taaccattct tgttggctaa
     2821 tataatatct tttatctgtg aaatacgatt tatttactgc aattaacaca tgaaaatgag
     2881 gattataatc atctcttttt ttattatatg taatctctaa cttacgaaca tatcccttta
     2941 taacactacc tacttttttt ctctttataa gttttctaaa agaattatta taacgtttta
     3001 tttcattttc taattcatca ctcattacat taggtgtagt caaagttaaa aagataaact
     3061 cctttttctc ttgctgctta atatattgca tcatcaaaga taaacccaat gcatcttttc
     3121 tagcttttct ccaagcacag acaggacaaa atcgattttt acaagaatta gctttatata
     3181 atttctgttt ttctaaagtt ttatcagcta caaaagacag aaatgtattg caatcttcaa
     3241 ctaaatccat ttgattctct ccaatatgac gtttaataaa tttctgaaat acttgatttc
     3301 tttgtttttt ctcagtatac ttttccatgt tataacacat aaaaacaact tagttttcac
     3361 aaactatgac aataaaaaaa gttgcttttt cccctttcta tgtatgtttt ttactattca
     3421 tttaaaacga tacattaata ggtacgaaaa agcaactttt tttgcgctta aaaccagtca
     3481 taccaataac ttaagggtaa ctagcctcgc cggcaatagt tacccttatt atcaagataa
     3541 gaaagaaaag gatttttcgc tacgctcaaa tcctttaaaa aaacacaaaa gaccacattt
     3601 tttaatgtgg tcttttattc ttcaactaaa gcacccatta gttcaacaaa cgaaaattgg
     3661 ataaagtggg atatttttaa aatatatatt tatgttacag taatattgac ttttaaaaaa
     3721 ggattgattc taatgaagaa agcagacaag taagcctcct aaattcactt tagataaaaa
     3781 tttaggaggc atatcaaatg aactttaata aaattgattt agacaattgg aagagaaaag
     3841 agatatttaa tcattatttg aaccaacaaa cgacttttag tataaccaca gaaattgata
     3901 ttagtgtttt ataccgaaac ataaaacaag aaggatataa attttaccct gcatttattt
     3961 tcttagtgac aagggtgata aactcaaata cagcttttag aactggttac aatagcgacg
     4021 gagagttagg ttattgggat aagttagagc cactttatac aatttttgat ggtgtatcta
     4081 aaacattctc tggtatttgg actcctgtaa agaatgactt caaagagttt tatgatttat
     4141 acctttctga tgtagagaaa tataatggtt cggggaaatt gtttcccaaa acacctatac
     4201 ctgaaaatgc tttttctctt tctattattc catggacttc atttactggg tttaacttaa
     4261 atatcaataa taatagtaat taccttctac ccattattac agcaggaaaa ttcattaata
     4321 aaggtaattc aatatattta ccgctatctt tacaggtaca tcattctgtt tgtgatggtt
     4381 atcatgcagg attgtttatg aactctattc aggaattgtc agataggcct aatgactggc
     4441 ttttataata tgagataatg ccgactgtac tttttacagt cggttttcta atgtcactaa
     4501 cctgccccgt tagttgaaga aggtttttat attacagctc cagatccata tccttctttt
     4561 tctgaaccga cttctccttt ttcgcttctt tattccaatt gctttattga cgttgagcct
     4621 cggaaccctt aacaatccca aaacttgtcg aatggtcggc ttaatagctc acgctatgcc
     4681 gacattcgtc tgcaagttta gttaagggtt cttctcaacg cacaataaat tttctcggca
     4741 taaatgcgtg gtctaatttt tatttttaat aaccttgata gcaaaaaatg ccattccaat
     4801 acaaaaccac atacctataa tcgataacca cataacagtc ataaaaccac tcctttttaa
     4861 caaactttat cacaagaaat atttaaattt taaatgcctt tattttgaat tttaaggggc
     4921 attttaaaga tttaggggta aatcatatag ttttatgcct aaaaacctac agaagcttgg
     4981 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5041 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5101 cattaattgc gttgcgctca ctgtccgctt tccagtcggg aaacctgtcg tgccagctgc
     5161 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5221 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5281 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5341 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5401 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5461 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5521 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5581 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5641 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5701 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5761 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5821 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5881 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5941 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6001 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6061 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6121 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6181 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6241 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6301 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6361 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6421 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6481 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6541 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6601 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6661 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6721 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6781 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6841 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6901 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6961 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7021 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7081 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7141 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     7201 ggccctttcg tctcgcgcgt ttc
//
