LOCUS       sequence-194293-        7121 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of fluorescent protein casette - cpEGFP 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 7121)
  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 7121)
  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-eGO-Matryoshka-LE-LP
FEATURES             Location/Qualifiers
     source          1..7121
                     /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"
     primer_bind     333..354
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             406..1113
                     /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     819..838
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1168..1189)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1429..1448)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(1622..1640)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1636..1683
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1858..1863,1864..1880,1881..1886))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1913..1931)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1999..2103
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2104..2172,2173..2964)
                     /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(2322..2341)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3135..3723
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3624..3643
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3877..3894
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3909..4048
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3974..3993)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4134..4156
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4150..4341)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5653..6735)
                     /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     6697..6716
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6736..6813)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7019..7038
                     /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 tctatatcaa ggccgacaag cagaagaacg gcatcaaggc gaacttccac
      181 atccgccaca acatcgagga cggcggcgtg cagctcgcct accactacca gcagaacacc
      241 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcgt gcagtccaaa
      301 ctttcgaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
      361 gccgggatca ctctcggcat ggacgagctg tacaagggcg gtaccatggt gagcaagggc
      421 gaggagaata acatggccat catcaaggag ttcatgcgct tcaaggtgcg catggagggc
      481 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
      541 tttcagaccg ttaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
      601 ttgtcccctc agttcaccta cggctccaag gcctacgtga agcaccccgc cgacatcccc
      661 gactacctca agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
      721 gacggcggcg tggtgaccgt gactcaggac tcctccctgc aggacggcga gttcatctac
      781 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc
      841 atgggcatgg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
      901 gacaagctca ggctgaagct gaaggacggc ggccactaca cctccgaggt caagaccacc
      961 tacaaggcca agaagcccgt gcagttgccc ggcgcctaca tcgtcgacat caagttggac
     1021 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     1081 cactccaccg gcggcatgga cgagctgtac aagggaggga gcatggtgag caagggcgag
     1141 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     1201 aagttcagcg tgtccggcga gggtgagggc gatgccacct acggcaagct gaccctgaag
     1261 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
     1321 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     1381 tccgccatgc ccgaaggcta catccaggag cgcaccatct tcttcaagga cgacggcaac
     1441 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     1501 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaacctg
     1561 ccgtaactcg aggatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     1621 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     1681 ttgctgaaag gaggaactat atccggatat cccgcaagag gcccggcagt accggcataa
     1741 ccaagcctat gcctacagca tccagggtga cggtgccgag gatgacgatg agcgcattgt
     1801 tagatttcat acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt
     1861 aaagcttatc gatgataagc tgtcaaacat gagaattctt gaagacgaaa gggcctcgtg
     1921 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     1981 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2041 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2101 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     2161 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     2221 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     2281 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     2341 tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     2401 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     2461 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     2521 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     2581 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     2641 atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     2701 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     2761 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     2821 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     2881 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     2941 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     3001 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     3061 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     3121 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3181 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     3241 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     3301 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3361 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3421 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3481 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3541 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3601 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3661 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3721 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     3781 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     3841 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     3901 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     3961 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     4021 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     4081 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     4141 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     4201 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     4261 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     4321 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     4381 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     4441 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     4501 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     4561 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     4621 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     4681 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     4741 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     4801 cacccgtggc caggacccaa cgctgcccga gatgcgccgc gtgcggctgc tggagatggc
     4861 ggacgcgatg gatatgttct gccaagggtt ggtttgcgca ttcacagttc tccgcaagaa
     4921 ttgattggct ccaattcttg gagtggtgaa tccgttagcg aggtgccgcc ggcttccatt
     4981 caggtcgagg tggcccggct ccatgcaccg cgacgcaacg cggggaggca gacaaggtat
     5041 agggcggcgc ctacaatcca tgccaacccg ttccatgtgc tcgccgaggc ggcataaatc
     5101 gccgtgacga tcagcggtcc aatgatcgaa gttaggctgg taagagccgc gagcgatcct
     5161 tgaagctgtc cctgatggtc gtcatctacc tgcctggaca gcatggcctg caacgcgggc
     5221 atcccgatgc cgccggaagc gagaagaatc ataatgggga aggccatcca gcctcgcgtc
     5281 gcgaacgcca gcaagacgta gcccagcgcg tcggccgcca tgccggcgat aatggcctgc
     5341 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     5401 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     5461 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     5521 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     5581 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     5641 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5701 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     5761 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     5821 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     5881 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     5941 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     6001 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     6061 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     6121 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     6181 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     6241 atcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     6301 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     6361 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     6421 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     6481 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     6541 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     6601 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     6661 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     6721 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     6781 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     6841 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     6901 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     6961 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     7021 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     7081 tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa t
//
