pTH100

LOCUS      pTH100               9969 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  pJB38-NWMN29-30 + SarA_P1-sGFP-Term.
ACCESSION   .
VERSION     .
KEYWORDS    pTH100
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9969)
  AUTHORS   de Jong NW, van der Horst T, van Strijp JA, Nijland R
  TITLE     Fluorescent reporters for markerless genomic integration in 
            Staphylococcus aureus.
  JOURNAL   Sci Rep. 2017 Mar 7;7:43889. doi: 10.1038/srep43889.
  PUBMED    28266573
REFERENCE   2  (bases 1 to 9969)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9969
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             1324..2037
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1354..1382)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1977..1997
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2044..2061
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2079..2095)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2079..2095)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     terminator      2104..2138
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     protein_bind    4965..4983
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    5054..5072
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     CDS             5097..5720
                     /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     complement(5170..5189)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(7711..7733)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    7819..7959
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     7874..7893
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(7974..7991)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8145..8733)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8225..8244)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(8904..9764)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     9527..9546
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9765..9869)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     9937..9955
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gaattcagtg gctacattcg aacatatcaa agcatacgat agaaaaacaa taagttatga
       61 tatgttacat gtaacaccac ctatgggtcc cttagatgta gtaaaagaaa gtacactttc
      121 agatagtgag ggttgggtag atgttaaccc aaccacatta cagcataaaa gctactctaa
      181 tgtatttgca cttggtgatg cttcaaatgt acctacttca aaaacaggcg cagctattcg
      241 taagcaagca cctatcgtcg ctaataattt attgcaagtg atgaataatc aaatgttaac
      301 gcatcattat gatggttata cttcatgccc tattgttact ggatataata ggttaatact
      361 tgcagagttt gattataata aaaatactaa agaaacaatg ccgtttaatc aggccaaaga
      421 acgtagaagt atgtatatat ttaagaaaga tttattacct aaaatgtatt ggtacggcat
      481 gctaaaagga ttaatataat aaagtacaga aaacaataaa tttttaatga aaaatctttt
      541 actataaaag attaagtatt taaatgacgt gtcagtgttg tgtttatatg tcgtgaattt
      601 ttagctctaa atagtataag attgaaaaag ttgttactgt tttaaatgat cacgatgaag
      661 tcattcaata agaatgatta tgaaaataga aacagcagta agatattttc taattgaaaa
      721 tcatctcact gctgtttttt aaaggtttat acctcatcct ctaaattatt taaaaataat
      781 taatggtatt tgagcacgtt tagcgacttt atgactgaca ttaccaattt ccatttcttg
      841 ccagatattc aaaccacgtg tactcaaaat gatagcttgg tatgtacctc caatagtaat
      901 ttcaataact ttgtctgttg aacactaaga gcaattttaa tttcataatg tgttgtaaac
      961 attttttttg attggagttt ttttctgagt taaacgatat atcgcgattg catgcctgca
     1021 ggtcgactct agaggatccg ctagcctgat atttttgact aaaccaaatg ctaacccaga
     1081 aatacaatca ctgtgtctaa tgaataattt gttttataaa cacttttttg tttacttctc
     1141 atttttaatt agttataatt aactaaataa tagagcatta aatatattta ataaaactta
     1201 tttaatgcaa aattatgact aacatatcta taataaataa agattagata tcaatatatt
     1261 atcgggcaaa tgtatcgagc aagatgcatc ggatccccgg gtaccttagg aggatgatta
     1321 tttatgagca aaggagaaga acttttcact ggagttgtcc caattcttgt tgaattagat
     1381 ggtgatgtta atgggcacaa attttctgtc cgtggagagg gtgaaggtga tgctacaaac
     1441 ggaaaactca cccttaaatt tatttgcact actggaaaac tacctgttcc gtggccaaca
     1501 cttgtcacta ctctgaccta tggtgttcaa tgcttttccc gttatccgga tcacatgaaa
     1561 cgtcatgact ttttcaagag tgccatgcct gaaggttatg tacaggaacg cactatatct
     1621 ttcaaagatg acgggaccta caagacgcgt gctgaagtca agtttgaagg tgataccctt
     1681 gttaatcgta tcgagttaaa gggtattgat tttaaagaag atggaaacat tcttggacac
     1741 aaactcgagt acaactttaa ctcacacaat gtatacatca cggcagacaa acaaaagaat
     1801 ggaatcaaag ctaacttcaa aattcgccac aacgttgaag atggttccgt tcaactagca
     1861 gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat
     1921 tacctgtcga cacaatctgt cctttcgaaa gatcctaacg aaaagcgtga ccacatggtc
     1981 cttcttgagt ttgtaactgc tgctgggatt acacatggca tggatgagct ctacaaagga
     2041 tcccaccacc accaccacca ctaagaattc gtaatcatgt catagctgtt tcctgtgtgt
     2101 taattcagaa cgctcggttg ccgccgggcg ttttttatgc agctcgcgat atatcctgat
     2161 gtatttttaa ttttgcacca tttccaaaag gataagtgac ataagtaaaa aggcatcatc
     2221 gggagttatc ctatcaggaa aaccaagata atacctaagt agaaagtgtt caatccgtgt
     2281 taaattggga aatatcatcc ataaacttta ttactcatac tataattcaa ttttaacgtc
     2341 ttcgtccatt tgggcttcaa attcatcgag tagtgctcgt gcttctgcaa ttgattgtgt
     2401 gttcatcaat tgatgtcgaa gttcgctagc gcctcttatg ccacgcacat agattttaaa
     2461 gaatctacgc aagctcttga attgtcgtat ttcatctttt tcatatttgt taaacaatga
     2521 taaatgcaat ctcaatagat ctaatagttc cttgcttgtg tgttcgcgtg gttctttttc
     2581 aaaagcgaat ggattgtgga aaatgcctct accaatcatg acgccatcaa tgccatattt
     2641 ttctgccagt tcaagtcctg tttttctatc gggaatatca ccgttaattg ttaacaatgt
     2701 atttggtgca atttcgtcac gtaaattttt aatagcttcg attaattccc aatgtgcatc
     2761 tactttactc atttctttac gtgtacgaag atgaatagat aaattggcaa tgtcttgttc
     2821 gaagacgtgc ttcaaccaat ctttccattc atcgatttca tagtagccaa ggcgtgtttt
     2881 aacacttacc ggaagcccac ctgctttagt cgcttgaata atttcggcag caacgtcagg
     2941 tcttaagatt aagccggaac ccttacggta cccggggatc ctctagagtc gacctgcagg
     3001 catgcaagct ctgatttcac tttttgcatt ctacaaactg cataactcat atgtaaatcg
     3061 ctccttttta ggtggcacaa atgtgaggca ttttcgctct ttccggcgag gctagttacc
     3121 cttaagttat tggtatgact ggttttaagc gcaaaaaaag ttgctttttc gtacctatta
     3181 atgtatcgtt agaaaaccga ctgtaaaaag tacagtcggc attatctcat attataaaag
     3241 ccagtcatta ggcctatctg acaattcctg aatagagttc ataaacaatc ctgcatgata
     3301 accatcacaa acagaatgat gtacctgtaa agatagcggt aaatatattg aattaccttt
     3361 attaatgaat tttcctgctg taataatggg tagaaggtaa ttactattat tattgatatt
     3421 taagttaaac ccagtaaatg aagtccatgg aataatagaa agagaaaaag cattttcagg
     3481 tataggtgtt ttgggaaaca atttccccga accattatat ttctctacat cagaaaggta
     3541 taaatcataa aactctttga agtcattctt tacaggagtc caaataccag agaatgtttt
     3601 agatacacca tcaaaaattg tataaagtgg ctctaactta tcccaataac ctaactctcc
     3661 gtcgctattg taaccagttc taaaagctgt atttgagttt atcacccttg tcactaagaa
     3721 aataaatgca gggtaaaatt tatatccttc ttgttttatg tttcggtata aaacactaat
     3781 atcaatttct gtggttatac taaaagtcgt ttgttggttc aaataatgat taaatatctc
     3841 ttttctcttc caattgtcta aatcaatttt attaaagttc atttgatatg cctcctaaat
     3901 ttttatctaa agtgaattta ggaggcttac ttgtctgctt tcttcattag aatcaatcct
     3961 tttttaaaag tcaatattac tgtaacataa atatatattt taaaaatatc ccactttatc
     4021 caattttcgt ttgttgaact aatgggtgct ttagttgaag aataaagacc acattaaaaa
     4081 atgtggtctt ttgtgttttt ttaaaggatt tgagcgtagc gaaaaatcct tttctttctt
     4141 atcttgataa taagggtaac tattgccggg atagactgta acattctcac gcataaaatc
     4201 ccctttcatt ttctaatgta aatctattac cttattatta attcaattcg ctcataatta
     4261 atcctttttc ttattacgca aaatggcccg atttaagcac accctttatt ccgttaatgc
     4321 gccatgacag ccatgataat tactaatact aggagaagtt aataaatacg ggtgatctaa
     4381 tgattcaatg attcaaaccc ttgtgaactt ctttagaaca aaagaggttc gtaacaagat
     4441 tttcttcaca ctagcaatgt tagtaatttt taaaataggg acttatatac cagctccagg
     4501 agtaaatcct gcagcttttg ataatcccca aggttctcaa ggtgccactg agttattaaa
     4561 tacttttggt ggcggagcct tgaaacgatt ttctattttt gcaatgggta ttgtacccta
     4621 catcactgca tcaatcgtaa tgcaattatt acaaatggat attgtcccta aattctcaga
     4681 atgggcaaaa caaggtgaag taggtagaag aaagttaaat aacgttactc gttatttagc
     4741 aatttcttta gcatttatcc aatctatagg tatggcattc caatttaata attatctcaa
     4801 aggtgcgctg attatcaatc agtcaattat gagttattta ttaatagcac tagttttgac
     4861 agcaggaact gctttcttaa tatggcttgg tgatcaaatc actcagttcg gtgttggtaa
     4921 tggtatttct attatcatat tcccatcaag cttattttaa ttatactcta tcaatgatag
     4981 agtgtcaata ttttttttag tttttcatga actcgatcga ggggatccaa ataaaaaact
     5041 agtttgacaa ataactctat caatgataga gtgtcaacaa aaaggaggaa ttaatgatgt
     5101 ctagattaga taaaagtaaa gtgattaaca gcgcattaga gctgcttaat gaggtcggaa
     5161 tcgaaggttt aacaacccgt aaactcgccc agaagctagg tgtagagcag cctacattgt
     5221 attggcatgt aaaaaataag cgggctttgc tcgacgcctt agccattgag atgttagata
     5281 ggcaccatac tcacttttgc cctttagaag gggaaagctg gcaagatttt ttacgtaata
     5341 acgctaaaag ttttagatgt gctttactaa gtcatcgcga tggagcaaaa gtacatttag
     5401 gtacacggcc tacagaaaaa cagtatgaaa ctctcgaaaa tcaattagcc tttttatgcc
     5461 aacaaggttt ttcactagag aatgcattat atgcactcag cgctgtgggg cattttactt
     5521 taggttgcgt attggaagat caagagcatc aagtcgctaa agaagaaagg gaaacaccta
     5581 ctactgatag tatgccgcca ttattacgac aagctatcga attatttgat caccaaggtg
     5641 cagagccagc cttcttattc ggccttgaat tgatcatatg cggattagaa aaacaactta
     5701 aatgtgaaag tgggtcttaa aagcagcata acctttttcc gtgatggtaa cttcacggta
     5761 accaagataa caaagaatac aagaaaatat ttacaaaaaa tcaatttaac aattccttaa
     5821 aacatgcagg aattgacgat ttaaacaata ttagctttga acaattctta tctcttttca
     5881 atagctataa attatttaat aagtaagtta agggatgcat aaactgcatc ccttaacttg
     5941 tttttcgtgt gcctattttt tgtgaatcga ttatgtcttt tgcgcagtcg gcttaaacca
     6001 gttttcgctg gtgcgaaaaa agagtgtctt gtgacaccta aattcaaaat ctatcggtca
     6061 gatttatacc gatttgattt tatatattct tgaataacat acgccgagtt atcacataaa
     6121 agcgggaacc aatcatcaaa tttaaacttc attgcataat ccattaaact cttaaattct
     6181 acgattcctt gttcatcaat aaactcaatc atttctttaa ttaatttata tctatctgtt
     6241 gttgttttct ttaataattc atcaacatct acaccgccat aaactatcat atcttctttt
     6301 tgatatttaa atttattagg atcgtccatg tgaagcatat atctcacaag acctttcaca
     6361 cttcctgcaa tctgcggaat agtcgcattc aattcttctg taattatttt tatctgttca
     6421 taagatttat taccctcata catcactaga atatgataat gctctttttt catcctatct
     6481 tctgtatcag tatccctatc atgtaatgga gacactacaa attgaatgtg taactctttt
     6541 aaatactcta accactcggc ttttgctgat tctggatata aaacaaatgt ccaattacgt
     6601 cctcttgaat ttttcttgtt ttcagtttct tttattacat tttcgctcat gatataataa
     6661 cggtgctaat acatttaaca aaatttagtc atagataggc agcatgccag tgctgtctat
     6721 ctttttttgt ttaaaatgca ccgtattcct cctttgcata tttttttatt agaataccgg
     6781 ttgcatctga tttgctaata ttatattttt ctttgattct atttaatatc tcattttctt
     6841 ctgttgtaag tcttaaagta acagcaactt ttttctcttc ttttctatct acaaccatca
     6901 ctgtacctcc caacatctgt ttttttcact ttaacataaa aaacaacctt ttaacattaa
     6961 aaacccaata tttatttatt tgtttggaca atggacaatg gacacctagg ggggaggtcg
     7021 tagtaccccc ctatgttttc tcccctaaat aaccccaaaa atctaagaaa aaaagacctc
     7081 aaaaaggtct ttaattaaca tctcaaattt cgcatttatt ccaatttcct ttttgcgtgt
     7141 gatgcgctgc gtccattaaa aatcctagag ctttgcaacc gaaagttaat agctgtcgct
     7201 actactttcg cttacgctct aagtatattt taaggactgt cacacgcaaa aagttttctc
     7261 ggcataaaag tacctctaca tctctaaatc gtctgtacgc tgtttctcac gctttctatc
     7321 gaccttctgg acattatcct gtacaacatc cataaactgt cccacacgct cgaatttgga
     7381 atcattaaag aatttctctt taagcctatt aaaccctttc tcaaacccag ggaaattcgc
     7441 cctcgcagca cgatataaag tcactgtact atcttgaaat ttctctgata cattcaactg
     7501 ctcattcaaa ctatcattct ctcgctttaa tttattaacc tctttacttt tttcgtgata
     7561 cccctctttc catgtattca ctacttcttt caaactctct ctacgttttt ttaattcttg
     7621 attttctgtg taatagtctg tgctcttaat attttcgtaa tcatcaacaa tccgttcctg
     7681 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     7741 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     7801 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac
     7861 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa
     7921 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
     7981 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     8041 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     8101 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     8161 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     8221 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     8281 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     8341 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     8401 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     8461 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     8521 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     8581 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     8641 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     8701 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     8761 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     8821 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     8881 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     8941 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     9001 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     9061 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     9121 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     9181 tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc
     9241 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     9301 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     9361 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     9421 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     9481 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca
     9541 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     9601 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     9661 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     9721 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     9781 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     9841 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     9901 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     9961 cgtcttcaa
//