LOCUS       sequence-194292-        7115 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of fluorescent protein casette - cpsfGFP with 
            nested LSSmOrange and LE N-term and LP C-term amino acid linkers.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7115)
  AUTHORS   Ast C, Foret J, Oltrogge LM, De Michele R, Kleist TJ, Ho CH, Frommer
            WB
  TITLE     Ratiometric Matryoshka biosensors from a nested cassette of green- 
            and orange-emitting fluorescent proteins.
  JOURNAL   Nat Commun. 2017 Sep 5;8(1):431. doi: 10.1038/s41467-017-00400-2.
  PUBMED    28874729
REFERENCE   2  (bases 1 to 7115)
  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"; journalName: "Nat 
            Commun."; date: "2017-09-5"; volume: "8(1):431. doi"; pages: " 
            10.1038/s41467-017-00400-2"
COMMENT     Sequence Label: pET15b-sfGO-Matryoshka-LE-LP
FEATURES             Location/Qualifiers
     source          1..7115
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             94..111
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             403..1110
                     /codon_start=1
                     /product="monomeric orange fluorescent protein with a large
                     Stokes shift (Shcherbakova et al., 2012)"
                     /label=LSSmOrange
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEG
                     FQTVKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGMEASSERMYPEDGALK
                     GEDKLRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVDIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     816..835
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1616..1634)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1630..1677
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1852..1857,1858..1874,1875..1880))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1907..1925)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1993..2097
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2098..2166,2167..2958)
                     /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     complement(2316..2335)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3129..3717
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3618..3637
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3871..3888
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3903..4042
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3968..3987)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4128..4150
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4144..4335)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5647..6729)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6691..6710
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6730..6807)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7013..7032
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg gggcatcatc atcatcatca tagcagcggc
      121 ctcgagaacg tgtatattac cgcggataaa cagaaaaacg gcattaaagc gaactttacc
      181 gtgcgccata acgtggaaga tggcagcgtg cagctggcgg atcattatca gcagaacacc
      241 ccgattggcg atggcccggt gctgctgccg gataaccatt atctgagcac ccagaccaag
      301 ctgagcaaag atccgaacga aaaacgcgat cacatggtgc tgctggaatt tgtgaccgca
      361 gcgggcatta cacacggcat ggatgaactg tatggcggca ccatggtgag caagggcgag
      421 gagaataaca tggccatcat caaggagttc atgcgcttca aggtgcgcat ggagggctcc
      481 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcttt
      541 cagaccgtta agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcttg
      601 tcccctcagt tcacctacgg ctccaaggcc tacgtgaagc accccgccga catccccgac
      661 tacctcaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
      721 ggcggcgtgg tgaccgtgac tcaggactcc tccctgcagg acggcgagtt catctacaag
      781 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg
      841 ggcatggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgaggac
      901 aagctcaggc tgaagctgaa ggacggcggc cactacacct ccgaggtcaa gaccacctac
      961 aaggccaaga agcccgtgca gttgcccggc gcctacatcg tcgacatcaa gttggacatc
     1021 acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac
     1081 tccaccggcg gcatggacga gctgtacaag ggcggcagcg cgagccaggg cgaagaactg
     1141 tttaccggcg tggtgccgat tctggtggaa ctggatggcg atgtgaacgg ccataaattt
     1201 agcgtgcgcg gcgaaggcga aggcgatgcg accattggca aactgaccct gaaatttatt
     1261 tccaccaccg gcaaactacc ggtgccgtgg ccgaccctgg tgaccacctt aacctatggc
     1321 gtgcagtgct ttagccgcta tccggatcat atgaaacgcc atgatttttt taaaagcgcg
     1381 atgccggaag gctatgtgca ggaacgcacc attagcttta aagatgatgg caaatataaa
     1441 acccgcgcgg tggtgaaatt tgaaggcgat accctggtga accgcattga actgaaaggc
     1501 accgatttta aagaagatgg caacattctg gggcataaac tggaatataa cctgccgtaa
     1561 ctcgaggatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     1621 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     1681 aaaggaggaa ctatatccgg atatcccgca agaggcccgg cagtaccggc ataaccaagc
     1741 ctatgcctac agcatccagg gtgacggtgc cgaggatgac gatgagcgca ttgttagatt
     1801 tcatacacgg tgcctgactg cgttagcaat ttaactgtga taaactaccg cattaaagct
     1861 tatcgatgat aagctgtcaa acatgagaat tcttgaagac gaaagggcct cgtgatacgc
     1921 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     1981 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2041 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     2101 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     2161 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     2221 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     2281 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     2341 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     2401 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     2461 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     2521 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     2581 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     2641 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     2701 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     2761 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     2821 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     2881 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     2941 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     3001 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     3061 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3121 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3181 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3241 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     3301 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3361 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3421 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3481 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3541 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3601 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3661 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3721 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3781 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     3841 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     3901 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     3961 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     4021 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     4081 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4141 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     4201 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     4261 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     4321 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     4381 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     4441 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     4501 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     4561 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     4621 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     4681 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     4741 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     4801 tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc
     4861 gatggatatg ttctgccaag ggttggtttg cgcattcaca gttctccgca agaattgatt
     4921 ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc
     4981 gaggtggccc ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg
     5041 gcgcctacaa tccatgccaa cccgttccat gtgctcgccg aggcggcata aatcgccgtg
     5101 acgatcagcg gtccaatgat cgaagttagg ctggtaagag ccgcgagcga tccttgaagc
     5161 tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc gggcatcccg
     5221 atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac
     5281 gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg
     5341 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     5401 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     5461 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     5521 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     5581 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     5641 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5701 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     5761 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     5821 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     5881 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     5941 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     6001 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     6061 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     6121 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     6181 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccatcttc
     6241 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     6301 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     6361 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     6421 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     6481 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     6541 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     6601 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     6661 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     6721 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     6781 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     6841 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     6901 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     6961 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     7021 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     7081 cggcgtagag gatcgagatc tcgatcccgc gaaat
//
