LOCUS       sequence-309094-        8778 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP and gRNA tag for protein and mRNA quantification, to attache to 
            the 3' end of the gene coding sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8778)
  AUTHORS   Brion C, Lutz SM, Albert FW
  TITLE     Simultaneous quantification of mRNA and protein in single cells 
            reveals post-transcriptional effects of genetic variation.
  JOURNAL   Elife. 2020 Nov 16;9. pii: 60645. doi: 10.7554/eLife.60645.
  PUBMED    33191917
REFERENCE   2  (bases 1 to 8778)
  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: "Elife."; 
            date: "2020-11-16"; volume: "9. pii: 60645. doi"; pages: " 
            10.7554/eLife.60645"
COMMENT     Sequence Label: Plas-gRNA-LEU2
FEATURES             Location/Qualifiers
     source          1..8778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             299..871
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      877..1074
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2501..2517)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2502..2518)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     rep_origin      complement(3301..3889)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3381..3400)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4060..4851,4852..4920))
                     /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     4683..4702
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4921..5025)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5735..5758
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             5782..6498
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(5815..5843)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     6438..6458
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     ncRNA           6552..6588
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        6609..6684
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           6689..6756
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(6792..6810)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        7067..7474
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             7475..8569
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
ORIGIN
        1 gctcaggggc atgatgtgac tgtcgcccgt acatttagcc catacatccc catgtataat
       61 catttgcatc catacatttt gatggccgca cggcgcgaag caaaaattac ggctcctcgc
      121 tgcagacctg cgagcaggga aacgctcccc tcacagacgc gttgaattgt ccccacgccg
      181 cgcccctgta gagaaatata aaaggttagg atttgccact gaggttcttc tttcatatac
      241 ttccttttaa aatcttgcta ggatacagtt ctcacatcac atccgaacat aaacaaccat
      301 gggtaccact cttgacgaca cggcttaccg gtaccgcacc agtgtcccgg gggacgccga
      361 ggccatcgag gcactggatg ggtccttcac caccgacacc gtcttccgcg tcaccgccac
      421 cggggacggc ttcaccctgc gggaggtgcc ggtggacccg cccctgacca aggtgttccc
      481 cgacgacgaa tcggacgacg aatcggacga cggggaggac ggcgacccgg actcccggac
      541 gttcgtcgcg tacggggacg acggcgacct ggcgggcttc gtggtcgtct cgtactccgg
      601 ctggaaccgc cggctgaccg tcgaggacat cgaggtcgcc ccggagcacc gggggcacgg
      661 ggtcgggcgc gcgttgatgg ggctcgcgac ggagttcgcc cgcgagcggg gcgccgggca
      721 cctctggctg gaggtcacca acgtcaacgc accggcgatc cacgcgtacc ggcggatggg
      781 gttcaccctc tgcggcctgg acaccgccct gtacgacggc accgcctcgg acggcgagca
      841 ggcgctctac atgagcatgc cctgccccta atcagtactg acaataaaaa gattcttgtt
      901 ttcaagaact tgtcatttgt atagtttttt tatattgtag ttgttctatt ttaatcaaat
      961 gttagcgtga tttatatttt ttttcgcctc gacatcatct gcccagatgc gaagttaagt
     1021 gcgcagaaag taatatcatg cgtcaatcgt atgtgaatgc tggtcgctat actgcctacc
     1081 caccgtactc gtcaattcca agggcatcgg taaacatctg ctcaaactcg aagtcggcca
     1141 tatccagagc gccgtagggg gcggagtcgt ggggggtaaa tcccggaccc ggggaatccc
     1201 cgtcccccaa catgtccaga tcgaaatcgt ctagcgcgtc ggcatgcgcc atcgccacgt
     1261 cctcgccgtc taagtggagc tccgatgaag tagagcccgc agtggccaag tggctttggt
     1321 ccgtctcctc cacggatgcc cctccacggc tagtgggcgc atgtaggcgg tgggcgtcca
     1381 gcatctccag cagcaggtca tagaggggca ccacgttctt gcacttcatg ctgtacagat
     1441 gctccatgcc tttgttactc atgtgcctga tgtgggagag gatgaggagg agctgggcca
     1501 gccgctggtg ctgctgctgc agggtcaggc ctgccttggc catcaggtgg atcaaagtgt
     1561 ctgtgatctt gtccaggact cggtggatat ggtccttctc ttccagagac ttcagggtgc
     1621 tggacagaaa tgtgtacact ccagaattaa gcaaaataat agatttgagg cacacaaact
     1681 cctctccctg cagattcatc atgcggaacc gagatgatgt agccagcagc atgtcgaaga
     1741 tctccaccat gccctctaca cattttccct ggttcctgtc caagagcaag ttaggagcaa
     1801 acagtagctt cactgggtgc tccatggagc gccagacgag accaatcatc aggatctcta
     1861 gccaggcaca ttctagaagg tggacctgat catggagggt caaatccaca aagcctggca
     1921 ccctcttcgc ccagttgatc atgtgaacca gctccctgtc tgccaggttg gtcagtaagc
     1981 ccatcatcga agcttcactg aagggtctgg taggatcata ctcggaatag agtatggggg
     2041 gctcagcatc caacaaggca ctgaccatct ggtcggccgt cagggacaag gccaggctgt
     2101 tcttcttaga gcgtttgatc atgagcgggc ttggccaaag gttggcagct ctcatgtctc
     2161 cagcagatgg atcctctaga gtcgacgcag ctgcaggtgt gcctccgcca cctgtatgga
     2221 ttttggtatg cctcttgcgt tcatcactcc tggcaaactt cctcccacaa atgtcacagg
     2281 caaaaggctt ctcgcctgtg tgggtgcgga tgtgggtggt aaggtggtca ctacgactga
     2341 agttacgcat gcagattcga cactggaagg gcttctgacc ggtatggatg cggatatggc
     2401 gggtaagctc atccgagcga gaaaagcggc gatcgcagga ctcgacaggg caagcatatg
     2461 ggcgggtacc catctttcag gaggcttgct tcaagcttat cgataccgtc gacctcgagg
     2521 gggggccctg ttaattcagt aaattttcga tcttgggaag aaaaaagcag taagcgtgaa
     2581 aaatctaaaa gctgatgtag tagaagatcc tattctttaa caaagattga ccttttcttt
     2641 ttcttcttgg tttgagtaga aaggggaagg aagaatacaa gagagaggaa aaaaaggaag
     2701 ataaaaagag agcgtgatat aaatgaatat atattaaaca agagagattg ggaaggaaag
     2761 gatcaaacaa acccaaaaat atttcaaaaa ggagagagag aggcgagttt ggtttcaaaa
     2821 cggtttattt atttatgcaa gaggacgtgg aagaaaaaga agaaggaaga aaaaaatttg
     2881 aaagaaaaaa acgcgtggcg ggtaaagaag aaaatggaaa atagaggccg ggtgacagag
     2941 aaatattgag ggttaattgg aaaatatgtt agggtgaggc atatgttttt aagggttttg
     3001 aggatccgat aaggaagaat gtaggttaaa tgttgtgcat taattgctgt ggcagcttac
     3061 ccgcttcccc acacattttg gtagtatctg tcctcttgtt attgttactg taattgtgta
     3121 tatatgttct cgcgtgtgtc ttatttactt atttagttat tatattatat gggtctgcaa
     3181 ggtagaggcg gcgcgggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     3241 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     3301 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     3361 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     3421 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     3481 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     3541 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     3601 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     3661 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     3721 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     3781 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     3841 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     3901 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     3961 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     4021 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     4081 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     4141 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     4201 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     4261 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     4321 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     4381 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     4441 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     4501 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     4561 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     4621 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     4681 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     4741 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     4801 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     4861 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     4921 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     4981 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     5041 agtgccacct gacgtcgagg caagctaaac agatctttat attgaatttt caaaaattct
     5101 tacttttttt ttggatggac gcaaagaagt ttaataatca tattacatgg cattaccacc
     5161 atatacatat ccatatacat atccatatct aatcttactt atatgttgtg gaaatgtaaa
     5221 gagccccatt atcttagcct aaaaaaacct tctctttgga actttcagta atacgcttaa
     5281 ctgctcattg ctatattgaa gtgcggccgc gtgggcgtgc gtgggcgggc gtgggcgtgc
     5341 gtgggcgggc gtgggcgtgc gtgggcgtct agaccgtgcg tcctcgtctt caccggtcgc
     5401 gttcctgaaa cgcagatgtg cctcgcgccg cactgctccg aacaataaag attctacaat
     5461 actagctttt atggttatga agaggaaaaa ttggcagtaa cctggcccca caaaccttca
     5521 aattaacgaa tcaaattaac aaccatagga tgataatgcg attagttttt tagccttatt
     5581 tctggggtaa ttaatcagcg aagcgatgat ttttgatcta ttaacagata tataaatgga
     5641 aaagctgcat aaccacttta actaatactt tcaacatttt cagtttgtat tacttcttat
     5701 tcaaatgtca taaaagtatc aacaaaaaat tgttaatata cctctatact ttaacgtcaa
     5761 ggagaaaaaa ctataggtac catggtgagt aaaggagaag aacttttcac tggagttgtc
     5821 ccaattcttg ttgaattaga tggtgatgtt aatgggcaca aattttctgt cagtggagag
     5881 ggtgaaggtg atgcaacata cggaaaactt acccttaaat ttatttgcac tactggaaaa
     5941 ctacctgttc catggccaac acttgtcact actttcactt atggtgttca atgcttttca
     6001 agatacccag atcatatgaa acggcatgac tttttcaaga gtgccatgcc cgaaggttat
     6061 gtacaggaaa gaactatatt tttcaaagat gacgggaact acaagacacg tgctgaagtc
     6121 aagtttgaag gtgataccct tgttaataga atcgagttaa aaggtattga ttttaaagaa
     6181 gatggaaaca ttcttggaca caaattggaa tacaactata actcacacaa tgtatacatc
     6241 atggcagaca aacaaaagaa tggaatcaaa gttaacttca aaattagaca caacattgaa
     6301 gatggaagcg ttcaactagc agaccattat caacaaaata ctccaattgg cgatggccct
     6361 gtccttttac cagacaacca ttacctgtcc acacaatctg ccctttcgaa agatcccaac
     6421 gaaaagagag accacatggt ccttcttgag tttgtaacag ctgctgggat tacacatggc
     6481 atggatgaac tatacaaata gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     6541 aaaaaatgta cctgatgagt ccgtgaggac gaaacgagta agctcgtcgt acatacagta
     6601 ggatcctagt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
     6661 gaaaaagtgg caccgagtcg gtgcttttgg ccggcatggt cccagcctcc tcgctggcgc
     6721 cggctgggca acatgcttcg gcatggcgaa tgggacactg acctaggaac tcgagtcatg
     6781 taattagtta tgtcacgctt acattcacgc ccttccccca catccgctct aaccgaaaag
     6841 gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta
     6901 ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca
     6961 tgtaacatta tactgaaaac cttgcttgag aaggctttaa tttgcggccc gacatggagg
     7021 cccagaatac cctccttgac agtcttgacg tgcgcagctc agggttaact gtgggaatac
     7081 tcaggtatcg taagatgcaa gagttcgaat ctcttagcaa ccattatttt tttcctcaac
     7141 ataacgagaa cacacagggg cgctatcgca cagaatcaaa ttcgatgact ggaaattttt
     7201 tgttaatttc agaggtcgcc tgacgcatat acctttttca actgaaaaat tgggagaaaa
     7261 aggaaaggtg agagcgccgg aaccggcttt tcatatagaa tagagaagcg ttcatgacta
     7321 aatgcttgca tcacaatact tgaagttgac aatattattt aaggacctat tgttttttcc
     7381 aataggtggt tagcaatcgt cttactttct aacttttctt accttttaca tttcagcaat
     7441 atatatatat atatttcaag gatataccat tctaatgtct gcccctaaga agatcgtcgt
     7501 tttgccaggt gaccacgttg gtcaagaaat cacagccgaa gccattaagg ttcttaaagc
     7561 tatttctgat gttcgttcca atgtcaagtt cgatttcgaa aatcatttaa ttggtggtgc
     7621 tgctatcgat gctacaggtg ttccacttcc agatgaggcg ctggaagcct ccaagaaggc
     7681 tgatgccgtt ttgttaggtg ctgtgggtgg tcctaaatgg ggtaccggta gtgttagacc
     7741 tgaacaaggt ttactaaaaa tccgtaaaga acttcaattg tacgccaact taagaccatg
     7801 taactttgca tccgactctc ttttagactt atctccaatc aagccacaat ttgctaaagg
     7861 tactgacttc gttgttgtca gagaattagt gggaggtatt tactttggta agagaaagga
     7921 agacgatggt gatggtgtcg cttgggatag tgaacaatac accgttccag aagtgcaaag
     7981 aatcacaaga atggccgctt tcatggccct acaacatgag ccaccattgc ctatttggtc
     8041 cttggataaa gctaatgttt tggcctcttc aagattatgg agaaaaactg tggaggaaac
     8101 catcaagaac gaattcccta cattgaaggt tcaacatcaa ttgattgatt ctgccgccat
     8161 gatcctagtt aagaacccaa cccacctaaa tggtattata atcaccagca acatgtttgg
     8221 tgatatcatc tccgatgaag cctccgttat cccaggttcc ttgggtttgt tgccatctgc
     8281 gtccttggcc tctttgccag acaagaacac cgcatttggt ttgtacgaac catgccacgg
     8341 ttctgctcca gatttgccaa agaataaggt caaccctatc gccactatct tgtctgctgc
     8401 aatgatgttg aaattgtcat tgaacttgcc tgaagaaggt aaggccattg aagatgcagt
     8461 taaaaaggtt ttggatgcag gtatcagaac tggtgattta ggtggttcca acagtaccac
     8521 cgaagtcggt gatgctgtcg ccgaagaagt taagaaaatc cttgcttaaa aagattctct
     8581 ttttttatga tatttgtaca taaactttat aaatgaaatt cataatagaa acgacacgaa
     8641 attacaaaat ggaatatgtt catagggtag acgaaactat atacgcaatc tacatacatt
     8701 tatcaagaag gagaaaaagg aggatgtaaa ggaatacagg taagcaaatt gatactaatg
     8761 gctcaacgtg ataaggaa
//
