LOCUS       sequence-407616-        8690 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of miRFP2-T2A-mtq2 in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8690)
  TITLE     Teusink lab plasmids
REFERENCE   2  (bases 1 to 8690)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pDRF1-GW miRFP2-T2A-mtq2
FEATURES             Location/Qualifiers
     source          1..8690
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    800..824
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1776..1829
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     regulatory      1845..1854
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1851..1853,1854..1856,1857..2570)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1896..1917)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2157..2176)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2504..2525
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2580..2604)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2666..2685)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2667..2685)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2833..3020
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2888..2912)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3032..3049)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3032..3048)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3041..3063)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3484..4830
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4941..5744)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(5745..5960)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5985..6003)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6071..6175
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6176..6244,6245..7036)
                     /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(6394..6413)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7207..7795
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7696..7715
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7949..7966
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8083..8104
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8119..8124,8125..8142,8143..8149)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8157..8173
                     /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)."
     primer_bind     8162..8184
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8181..8197
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8181..8197
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(8227..8682)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8364..8385)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8576..8595
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 caagcttcct gaaacggaga aacataaaca ggcattgctg ggatcaccca tacatcactc
       61 tgttttgcct gaccttttcc ggtaatttga aaacaaaccc ggtctcgaag cggagatccg
      121 gcgataatta ccgcagaaat aaacccatac acgagacgta gaaccagccg cacatggccg
      181 gagaaactcc tgcgagaatt tcgtaaactc gcgcgcattg catctgtatt tcctaatgcg
      241 gcacttccag gcctcgatcg agaccgttta tccattgctt ttttgttgtc tttttccctc
      301 gttcacagaa agtctgaaga agctatagta gaactatgag ctttttttgt ttctgttttc
      361 cttttttttt tttttacctc tgtggaaatt gttactctca cactctttag ttcgtttgtt
      421 tgttttgttt attccaatta tgaccggtga cgaaacgtgg tcgatggtgg gtaccgctta
      481 tgctcccctc cattagtttc gattatataa aaaggccaaa tattgtatta ttttcaaatg
      541 tcctatcatt atcgtctaac atctaatttc tcttaaattt tttctctttc tttcctataa
      601 caccaatagt gaaaatcttt ttttcttcta tatctacaaa aacttttttt ttctatcaac
      661 ctcgttgata aattttttct ttaacaatcg ttaataatta attaattgga aaataaccat
      721 tttttctctc ttttatacac acattcaaaa gaaagaaaaa aaatataccc cagcctcgac
      781 tagtgaattc gcggccatca caagtttgta caaaaaagca ggctgctagc atggtcgcag
      841 gacacgcaag tggaagcccc gactttggaa ccgccagtcc gagcgactgt gagcacgaag
      901 aaatccatct ggccggatcc atccagccac acggaacact gctggtggtc tccgagcctg
      961 accatagaat aattcaggca tctgccaacg ccgctgaatt cctgaatctg ggaagtgtgc
     1021 tgggaatccc cctggctgag atcgacgggg atctactgat caagattctg ccccacctgg
     1081 accttaccgc agaaggaatg ccagtggccg tcaggtgccg cattggaaac ccctccacag
     1141 agtacgacgg actgatgcat cgaccacctg aaggaggact gatcattgag ctggaacggg
     1201 ctgggccatc cattgatctg agcggaacac tggctcccgc actggagcga attcgaactg
     1261 caggatccct gagggctctg tgcgacgata ctgtactgct gttccagcag tgtaccggct
     1321 acgaccgagt gatggtctat cggttcgatg aacgggggca cggagaggtg tattcagaaa
     1381 ttcatgtgac tggactggag agctactttg gcaacaggta tcctagctcc ctcgtgccac
     1441 agatggcccg gcggctgtac gtgagacaga gggtgcgcgt cctggtagat gtcacctatc
     1501 agcctgtgcc actggagcca agactgagtc ccctgactgg cagggacctg gatatgtcag
     1561 ggtgttttct gcgctctatg agtcctactc acctgcagtt cctgaagaac atgggcgtgc
     1621 gggcaacact ggtggtcagc ctggtggtcg gaggcaaact gtgggggctg gtgatctgcc
     1681 accattatct gccacgcttc atccattttg agctgcgagc aatttgtgtg ctgctggccg
     1741 aagctatcgc aacccgcatt acagccctgt ccggagaggg cagaggaagt cttctaacat
     1801 gcggtgacgt ggaggagaat cccggccctg gggatccacc ggtcgccacc atggtgagca
     1861 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     1921 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     1981 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     2041 ccctgtcctg gggcgtgcag tgcttcgccc gctaccccga ccacatgaag cagcacgact
     2101 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     2161 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     2221 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     2281 acaactactt tagcgacaac gtctatatca ccgccgacaa gcagaagaac ggcatcaagg
     2341 ccaacttcaa gatccgccac aacatcgagg acggcggcgt gcagctcgcc gaccactacc
     2401 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     2461 cccagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     2521 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa agcggccgca
     2581 cccagctttc ttgtacaaag tggtgtagtt aattcatgat ggccgctgca ggtcgacctc
     2641 gagggggggc ccggtaccca attcgcccta tagtgagtcg tattacgcgc ggatccagct
     2701 ttggacttct tcgccagagg tttggtcaag tctccaatca aggttgtcgg cttgtctacc
     2761 ttgccagaaa tttacgaaaa gatggaaaag ggtcaaatcg ttggtagata cgttgttgac
     2821 acttctaaat aagcgaattt cttatgattt atgattttta ttattaaata agttataaaa
     2881 aaaataagtg tatacaaatt ttaaagtgac tcttaggttt taaaacgaaa attcttattc
     2941 ttgagtaact ctttcctgta ggtcaggttg ctttctcagg tatagcatga ggtcgctctt
     3001 attgaccaca cctctaccgg catgccaatt cactggccgt cgttttacaa cgtcgtgact
     3061 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     3121 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     3181 gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3241 taatcggatc gtacttgtta cccatcattg aattttgaac atccgaacct gggagttttc
     3301 cctgaaacag atagtatatt tgaacctgta taataatata tagtctagcg ctttacggaa
     3361 gacaatgtat gtatttcggt tcctggagaa actattgcat ctattgcata ggtaatcttg
     3421 cacgtcgcat ccccggttca ttttctgcgt ttccatcttg cacttcaata gcatatcttt
     3481 gttaacgaag catctgtgct tcattttgta gaacaaaaat gcaacgcgag agcgctaatt
     3541 tttcaaacaa agaatctgag ctgcattttt acagaacaga aatgcaacgc gaaagcgcta
     3601 ttttaccaac gaagaatctg tgcttcattt ttgtaaaaca aaaatgcaac gcgagagcgc
     3661 taatttttca aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgagag
     3721 cgctatttta ccaacaaaga atctatactt cttttttgtt ctacaaaaat gcatcccgag
     3781 agcgctattt ttctaacaaa gcatcttaga ttactttttt tctcctttgt gcgctctata
     3841 atgcagtctc ttgataactt tttgcactgt aggtccgtta aggttagaag aaggctactt
     3901 tggtgtctat tttctcttcc ataaaaaaag cctgactcca cttcccgcgt ttactgatta
     3961 ctagcgaagc tgcgggtgca ttttttcaag ataaaggcat ccccgattat attctatacc
     4021 gatgtggatt gcgcatactt tgtgaacaga aagtgatagc gttgatgatt cttcattggt
     4081 cagaaaatta tgaacggttt cttctatttt gtctctatat actacgtata ggaaatgttt
     4141 acattttcgt attgttttcg attcactcta tgaatagttc ttactacaat ttttttgtct
     4201 aaagagtaat actagagata aacataaaaa atgtagaggt cgagtttaga tgcaagttca
     4261 aggagcgaaa ggtggatggg taggttatat agggatatag cacagagata tatagcaaag
     4321 agatactttt gagcaatgtt tgtggaagcg gtattcgcaa tattttagta gctcgttaca
     4381 gtccggtgcg tttttggttt tttgaaagtg cgtcttcaga gcgcttttgg ttttcaaaag
     4441 cgctctgaag ttcctatact ttctagctag agaataggaa cttcggaata ggaacttcaa
     4501 agcgtttccg aaaacgagcg cttccgaaaa tgcaacgcga gctgcgcaca tacagctcac
     4561 tgttcacgtc gcacctatat ctgcgtgttg cctgtatata tatatacatg agaagaacgg
     4621 catagtgcgt gtttatgctt aaatgcgtac ttatatgcgt ctatttatgt aggatgaaag
     4681 gtagtctagt acctcctgtg atattatccc attccatgcg gggtatcgta tgcttccttc
     4741 agcactaccc tttagctgtt ctatatgctg ccactcctca attggattag tctcatcctt
     4801 caatgctatc atttcctttg atattggatc gatccgatga taagctgtca aacatgagaa
     4861 ttgggtaata actgatataa ttaaattgaa gctctaattt gtgagtttag tatacatgca
     4921 tttacttata atacagtttt ttagttttgc tggccgcatc ttctcaaata tgcttcccag
     4981 cctgcttttc tgtaacgttc accctctacc ttagcatccc ttccctttgc aaatagtcct
     5041 cttccaacaa taataatgtc agatcctgta gagaccacat catccacggt tctatactgt
     5101 tgacccaatg cgtctccctt gtcatctaaa cccacaccgg gtgtcataat caaccaatcg
     5161 taaccttcat ctcttccacc catgtctctt tgagcaataa agccgataac aaaatctttg
     5221 tcgctcttcg caatgtcaac agtaccctta gtatattctc cagtagatag ggagcccttg
     5281 catgacaatt ctgctaacat caaaaggcct ctaggttcct ttgttacttc ttctgccgcc
     5341 tgcttcaaac cgctaacaat acctgggccc accacaccgt gtgcattcgt aatgtctgcc
     5401 cattctgcta ttctgtatac acccgcagag tactgcaatt tgactgtatt accaatgtca
     5461 gcaaattttc tgtcttcgaa gagtaaaaaa ttgtacttgg cggataatgc ctttagcggc
     5521 ttaactgtgc cctccatgga aaaatcagtc aagatatcca catgtgtttt tagtaaacaa
     5581 attttgggac ctaatgcttc aactaactcc agtaattcct tggtggtacg aacatccaat
     5641 gaagcacaca agtttgtttg cttttcgtgc atgatattaa atagcttggc agcaacagga
     5701 ctaggatgag tagcagcacg ttccttatat gtagctttcg acatgattta tcttcgtttc
     5761 ctgcatgttt ttgttctgtg cagttgggtt aagaatactg ggcaatttca tgtttcttca
     5821 acactacata tgcgtatata taccaatcta agtctgtgct ccttccttcg ttcttccttc
     5881 tgttcggaga ttaccgaatc aaaaaaattt caaggaaacc gaaatcaaaa aaaagaataa
     5941 aaaaaaaatg atgaattgaa aagctaattc ttgaagacga aagggcctcg tgatacgcct
     6001 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     6061 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     6121 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     6181 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     6241 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     6301 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     6361 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     6421 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     6481 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     6541 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     6601 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     6661 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6721 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6781 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6841 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     6901 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     6961 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     7021 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     7081 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     7141 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     7201 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     7261 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     7321 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     7381 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     7441 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     7501 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     7561 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     7621 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     7681 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     7741 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     7801 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     7861 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     7921 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     7981 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     8041 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     8101 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     8161 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agcttaccgc
     8221 atcaggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     8281 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     8341 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     8401 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     8461 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     8521 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     8581 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     8641 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc
//
