LOCUS       sequence-309102-        7056 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP gene and gRNA under Z3EV expression control (estradiol sensor), 
            with Z3EV transcription factor to insert in the genome..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7056)
  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 7056)
  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-Z3EV
FEATURES             Location/Qualifiers
     source          1..7056
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        32..136
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(137..205,206..997)
                     /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     complement(355..374)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1168..1756
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1657..1676
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2539..2555
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2540..2556
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     gene            complement(3983..5102)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(3983..4180)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(4186..4758)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(4759..5102)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     primer_bind     5303..5321
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     ncRNA           complement(5357..5424)
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     misc_RNA        complement(5429..5504)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           complement(5525..5561)
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     CDS             complement(5615..6331)
                     /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(5655..5675)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     6270..6298
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     complement(6355..6378)
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
ORIGIN
        1 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
       61 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
      121 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
      181 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
      241 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
      301 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
      361 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
      421 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
      481 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
      541 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
      601 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
      661 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
      721 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
      781 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
      841 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
      901 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
      961 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     1021 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     1081 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     1141 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     1201 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     1261 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     1321 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     1381 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     1441 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     1501 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     1561 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     1621 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     1681 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     1741 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     1801 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     1861 ccgcgccgcc tctaccttgc agacccatat aatataataa ctaaataagt aaataagaca
     1921 cacgcgagaa catatataca caattacagt aacaataaca agaggacaga tactaccaaa
     1981 atgtgtgggg aagcgggtaa gctgccacag caattaatgc acaacattta acctacattc
     2041 ttccttatcg gatcctcaaa acccttaaaa acatatgcct caccctaaca tattttccaa
     2101 ttaaccctca atatttctct gtcacccggc ctctattttc cattttcttc tttacccgcc
     2161 acgcgttttt ttctttcaaa tttttttctt ccttcttctt tttcttccac gtcctcttgc
     2221 ataaataaat aaaccgtttt gaaaccaaac tcgcctctct ctctcctttt tgaaatattt
     2281 ttgggtttgt ttgatccttt ccttcccaat ctctcttgtt taatatatat tcatttatat
     2341 cacgctctct ttttatcttc ctttttttcc tctctcttgt attcttcctt cccctttcta
     2401 ctcaaaccaa gaagaaaaag aaaaggtcaa tctttgttaa agaataggat cttctactac
     2461 atcagctttt agatttttca cgcttactgc ttttttcttc ccaagatcga aaatttactg
     2521 aattaacagg gccccccctc gaggtcgacg gtatcgataa gcttgaagca agcctcctga
     2581 aagatgggta cccgcccata tgcttgccct gtcgagtcct gcgatcgccg cttttctcgc
     2641 tcggatgagc ttacccgcca tatccgcatc cataccggtc agaagccctt ccagtgtcga
     2701 atctgcatgc gtaacttcag tcgtagtgac caccttacca cccacatccg cacccacaca
     2761 ggcgagaagc cttttgcctg tgacatttgt gggaggaagt ttgccaggag tgatgaacgc
     2821 aagaggcata ccaaaatcca tacaggtggc ggaggcacac ctgcagctgc gtcgactcta
     2881 gaggatccat ctgctggaga catgagagct gccaaccttt ggccaagccc gctcatgatc
     2941 aaacgctcta agaagaacag cctggccttg tccctgacgg ccgaccagat ggtcagtgcc
     3001 ttgttggatg ctgagccccc catactctat tccgagtatg atcctaccag acccttcagt
     3061 gaagcttcga tgatgggctt actgaccaac ctggcagaca gggagctggt tcacatgatc
     3121 aactgggcga agagggtgcc aggctttgtg gatttgaccc tccatgatca ggtccacctt
     3181 ctagaatgtg cctggctaga gatcctgatg attggtctcg tctggcgctc catggagcac
     3241 ccagtgaagc tactgtttgc tcctaacttg ctcttggaca ggaaccaggg aaaatgtgta
     3301 gagggcatgg tggagatctt cgacatgctg ctggctacat catctcggtt ccgcatgatg
     3361 aatctgcagg gagaggagtt tgtgtgcctc aaatctatta ttttgcttaa ttctggagtg
     3421 tacacatttc tgtccagcac cctgaagtct ctggaagaga aggaccatat ccaccgagtc
     3481 ctggacaaga tcacagacac tttgatccac ctgatggcca aggcaggcct gaccctgcag
     3541 cagcagcacc agcggctggc ccagctcctc ctcatcctct cccacatcag gcacatgagt
     3601 aacaaaggca tggagcatct gtacagcatg aagtgcaaga acgtggtgcc cctctatgac
     3661 ctgctgctgg agatgctgga cgcccaccgc ctacatgcgc ccactagccg tggaggggca
     3721 tccgtggagg agacggacca aagccacttg gccactgcgg gctctacttc atcggagctc
     3781 cacttagacg gcgaggacgt ggcgatggcg catgccgacg cgctagacga tttcgatctg
     3841 gacatgttgg gggacgggga ttccccgggt ccgggattta ccccccacga ctccgccccc
     3901 tacggcgctc tggatatggc cgacttcgag tttgagcaga tgtttaccga tgcccttgga
     3961 attgacgagt acggtgggta ggcagtatag cgaccagcat tcacatacga ttgacgcatg
     4021 atattacttt ctgcgcactt aacttcgcat ctgggcagat gatgtcgagg cgaaaaaaaa
     4081 tataaatcac gctaacattt gattaaaata gaacaactac aatataaaaa aactatacaa
     4141 atgacaagtt cttgaaaaca agaatctttt tattgtcagt actgattagg ggcagggcat
     4201 gctcatgtag agcgcctgct cgccgtccga ggcggtgccg tcgtacaggg cggtgtccag
     4261 gccgcagagg gtgaacccca tccgccggta cgcgtggatc gccggtgcgt tgacgttggt
     4321 gacctccagc cagaggtgcc cggcgccccg ctcgcgggcg aactccgtcg cgagccccat
     4381 caacgcgcgc ccgaccccgt gcccccggtg ctccggggcg acctcgatgt cctcgacggt
     4441 cagccggcgg ttccagccgg agtacgagac gaccacgaag cccgccaggt cgccgtcgtc
     4501 cccgtacgcg acgaacgtcc gggagtccgg gtcgccgtcc tccccgtcgt ccgattcgtc
     4561 gtccgattcg tcgtcgggga acaccttggt caggggcggg tccaccggca cctcccgcag
     4621 ggtgaagccg tccccggtgg cggtgacgcg gaagacggtg tcggtggtga aggacccatc
     4681 cagtgcctcg atggcctcgg cgtcccccgg gacactggtg cggtaccggt aagccgtgtc
     4741 gtcaagagtg gtacccatgg ttgtttatgt tcggatgtga tgtgagaact gtatcctagc
     4801 aagattttaa aaggaagtat atgaaagaag aacctcagtg gcaaatccta accttttata
     4861 tttctctaca ggggcgcggc gtggggacaa ttcaacgcgt ctgtgagggg agcgtttccc
     4921 tgctcgcagg tctgcagcga ggagccgtaa tttttgcttc gcgccgtgcg gccatcaaaa
     4981 tgtatggatg caaatgatta tacatgggga tgtatgggct aaatgtacgg gcgacagtca
     5041 catcatgccc ctgagctgcg cacgtcaaga ctgtcaagga gggtattctg ggcctccatg
     5101 tcgggccgca aattaaagcc ttctcaagca aggttttcag tataatgtta catgcgtaca
     5161 cgcgtctgta cagaaaaaaa agaaaaattt gaaatataaa taacgttctt aatactaaca
     5221 taactataaa aaaataaata gggacctaga cttcaggttg tctaactcct tccttttcgg
     5281 ttagagcgga tgtgggggaa gggcgtgaat gtaagcgtga cataactaat tacatgactc
     5341 gagttcctag gtcagtgtcc cattcgccat gccgaagcat gttgcccagc cggcgccagc
     5401 gaggaggctg ggaccatgcc ggccaaaagc accgactcgg tgccactttt tcaagttgat
     5461 aacggactag ccttatttta acttgctatt tctagctcta aaactaggat cctactgtat
     5521 gtacgacgag cttactcgtt tcgtcctcac ggactcatca ggtacatttt tttttttttt
     5581 tttttttttt tttttttttt tttttttttt tctatttgta tagttcatcc atgccatgtg
     5641 taatcccagc agctgttaca aactcaagaa ggaccatgtg gtctctcttt tcgttgggat
     5701 ctttcgaaag ggcagattgt gtggacaggt aatggttgtc tggtaaaagg acagggccat
     5761 cgccaattgg agtattttgt tgataatggt ctgctagttg aacgcttcca tcttcaatgt
     5821 tgtgtctaat tttgaagtta actttgattc cattcttttg tttgtctgcc atgatgtata
     5881 cattgtgtga gttatagttg tattccaatt tgtgtccaag aatgtttcca tcttctttaa
     5941 aatcaatacc ttttaactcg attctattaa caagggtatc accttcaaac ttgacttcag
     6001 cacgtgtctt gtagttcccg tcatctttga aaaatatagt tctttcctgt acataacctt
     6061 cgggcatggc actcttgaaa aagtcatgcc gtttcatatg atctgggtat cttgaaaagc
     6121 attgaacacc ataagtgaaa gtagtgacaa gtgttggcca tggaacaggt agttttccag
     6181 tagtgcaaat aaatttaagg gtaagttttc cgtatgttgc atcaccttca ccctctccac
     6241 tgacagaaaa tttgtgccca ttaacatcac catctaattc aacaagaatt gggacaactc
     6301 cagtgaaaag ttcttctcct ttactcacca tggtacctat agttttttct ccttgacgtt
     6361 aaagtataga ggtatattaa caattttttg ttgatacttt tatgacattt gaataagaag
     6421 taatacaaac tgaaaatgtt gaaagtatta gttaaagtgg ttatgcagct tttccattta
     6481 tatatctgtt aatagatcaa aaatcatcgc ttcgctgatt aattacccca gaaataaggc
     6541 taaaaaacta atcgcattat catcctatgg ttgttaattt gattcgttaa tttgaaggtt
     6601 tgtggggcca ggttactgcc aatttttcct cttcataacc ataaaagcta gtattgtaga
     6661 atctttattg ttcggagcag tgcggcgcga ggcacatctg cgtttcagga acgcgaccgg
     6721 tgaagacgag gacgcacggt ctagacgccc acgcacgccc acgcccgccc acgcacgccc
     6781 acgcccgccc acgcacgccc acgcggccgc acttcaatat agcaatgagc agttaagcgt
     6841 attactgaaa gttccaaaga gaaggttttt ttaggctaag ataatggggc tctttacatt
     6901 tccacaacat ataagtaaga ttagatatgg atatgtatat ggatatgtat atggtggtaa
     6961 tgccatgtaa tatgattatt aaacttcttt gcgtccatcc aaaaaaaaag taagaatttt
     7021 tgaaaattca atataaagat ctgtttagct tgcctc
//
