LOCUS       sequence-64532-e        6561 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6561)
  AUTHORS   Hocine S, Raymond P, Zenklusen D, Chao JA, Singer RH
  TITLE     Single-molecule analysis of gene expression using two-color RNA 
            labeling in live yeast.
  JOURNAL   Nat Methods. 2013 Feb;10(2):119-21. doi: 10.1038/nmeth.2305. Epub 
            2012 Dec 23.
  PUBMED    23263691
REFERENCE   2  (bases 1 to 6561)
  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"; doi: 
            "10.1038/nmeth.2305"; journalName: "Nat Methods"; date: "2013-02"; 
            volume: "10"; issue: "2"; pages: "119-21"
COMMENT     Sequence Label: pSH100 (YCplac33 MET25pro MCP-mCherry)
FEATURES             Location/Qualifiers
     source          1..6561
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        65..414
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     regulatory      802..811
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             808..1518
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(953..971)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1221..1240
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1521..1537)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1522..1538)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      1540..1787
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1556..1574)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(1805..1824)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1806..1824)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1834..1851)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1834..1850)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1843..1865)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1995..2450
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2082..2101)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2292..2313
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2581..3384)
                     /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(3385..3605)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(3631..3650)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3750..3772
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3810..3828)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    3870..4373
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        4410..4514
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4515..4583,4584..5375)
                     /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(4733..4752)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5546..6134
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6035..6054
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6288..6305
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(6458..6463,6464..6481,6482..6488)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6496..6512
                     /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     6501..6523
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6520..6536
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6520..6536
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     join(6555..6561,1..14)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        join(6557..6561,1..14)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 accctcacta aagggaacaa aagctggagc tccggatgca agggttcgaa tcccttagct
       61 ctcattattt tttgcttttt ctcttgaggt cacatgatcg caaaatggca aatggcacgt
      121 gaagctgtcg atattgggga actgtggtgg ttggcaaatg actaattaag ttagtcaagg
      181 cgccatcctc atgaaaactg tgtaacataa taaccgaagt gtcgaaaagg tggcaccttg
      241 tccaattgaa cacgctcgat gaaaaaaata agatatatat aaggttaagt aaagcgtctg
      301 ttagaaagga agtttttcct ttttcttgct ctcttgtctt ttcatctact atttccttcg
      361 tgtaatacag ggtcgtcaga tacatagata caattctatt acccccatcc atactctaga
      421 atgctagccg ttaaaatggc ttctaacttt actcagttcg ttctcgtcga caatggcgga
      481 actggcgacg tgactgtcgc cccaagcaac ttcgctaacg ggatcgctga atggatcagc
      541 tctaactcgc gttcacaggc ttacaaagta acctgtagcg ttcgtcagag ctctgcgcag
      601 aatcgcaaat acaccatcaa agtcgaggtg cctaaaggcg cctggcgttc gtacttaaat
      661 atggaactaa ccattccaat tttcgccacg aactccgact gcgagcttat tgttaaggca
      721 atgcaaggtc tcctaaaaga tggaaacccg attccctcag caatcgcagc aaactccggc
      781 atctacgcgg atccaccggt cgccaccatg gtgagcaagg gcgaggagga taacatggcc
      841 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
      901 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
      961 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
     1021 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
     1081 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     1141 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     1201 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
     1261 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag
     1321 ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc
     1381 gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag
     1441 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     1501 gacgagctgt acaagtaaat cgataccgtc gacctcgagt catgtaatta gttatgtcac
     1561 gcttacattc acgccctccc cccacatccg ctctaaccga aaaggaagga gttagacaac
     1621 ctgaagtcta ggtccctatt tattttttta tagttatgtt agtattaaga acgttattta
     1681 tatttcaaat ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac attatactga
     1741 aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt aatttgcggc cggtacccaa
     1801 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga
     1861 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     1921 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     1981 tggcgaatgg cgcgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2041 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2101 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2161 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2221 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2281 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2341 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2401 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt cctgatgcgg
     2461 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tagggtaata actgatataa
     2521 ttaaattgaa gctctaattt gtgagtttag tatacatgca tttacttata atacagtttt
     2581 ttagttttgc tggccgcatc ttctcaaata tgcttcccag cctgcttttc tgtaacgttc
     2641 accctctacc ttagcatccc ttccctttgc aaatagtcct cttccaacaa taataatgtc
     2701 agatcctgta gagaccacat catccacggt tctatactgt tgacccaatg cgtctccctt
     2761 gtcatctaaa cccacaccgg gtgtcataat caaccaatcg taaccttcat ctcttccacc
     2821 catgtctctt tgagcaataa agccgataac aaaatctttg tcgctcttcg caatgtcaac
     2881 agtaccctta gtatattctc cagtagatag ggagcccttg catgacaatt ctgctaacat
     2941 caaaaggcct ctaggttcct ttgttacttc ttctgccgcc tgcttcaaac cgctaacaat
     3001 acctgggccc accacaccgt gtgcattcgt aatgtctgcc cattctgcta ttctgtatac
     3061 acccgcagag tactgcaatt tgactgtatt accaatgtca gcaaattttc tgtcttcgaa
     3121 gagtaaaaaa ttgtacttgg cggataatgc ctttagcggc ttaactgtgc cctccatgga
     3181 aaaatcagtc aagatatcca catgtgtttt tagtaaacaa attttgggac ctaatgcttc
     3241 aactaactcc agtaattcct tggtggtacg aacatccaat gaagcacaca agtttgtttg
     3301 cttttcgtgc atgatattaa atagcttggc agcaacagga ctaggatgag tagcagcacg
     3361 ttccttatat gtagctttcg acatgattta tcttcgtttc ctgcaggttt ttgttctgtg
     3421 cagttgggtt aagaatactg ggcaatttca tgtttcttca acactacata tgcgtatata
     3481 taccaatcta agtctgtgct ccttccttcg ttcttccttc tgttcggaga ttaccgaatc
     3541 aaaaaaattt caaagaaacc gaaatcaaaa aaaagaataa aaaaaaaatg atgaattgaa
     3601 ttgaaaagct gtggtatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3661 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     3721 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     3781 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     3841 tgtcatgata ataatggttt cttaggacgg atcgcttgcc tgtaacttac acgcgcctcg
     3901 tatcttttaa tgatggaata atttgggaat ttactctgtg tttatttatt tttatgtttt
     3961 gtatttggat tttagaaagt aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa
     4021 aataaacaaa ggtttaaaaa atttcaacaa aaagcgtact ttacatatat atttattaga
     4081 caagaaaagc agattaaata gatatacatt cgattaacga taagtaaaat gtaaaatcac
     4141 aggattttcg tgtgtggtct tctacacaga caagatgaaa caattcggca ttaatacctg
     4201 agagcaggaa gagcaagata aaaggtagta tttgttggcg atccccctag agtcttttac
     4261 atcttcggaa aacaaaaact attttttctt taatttcttt ttttactttc tatttttaat
     4321 ttatatattt atattaaaaa atttaaatta taattatttt tatagcacgt gatgaaaagg
     4381 acccaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4441 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4501 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4561 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4621 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     4681 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     4741 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     4801 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     4861 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     4921 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     4981 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5041 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5101 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     5161 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5221 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5281 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5341 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5401 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5461 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5521 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5581 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5641 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     5701 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5761 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5821 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5881 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5941 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6001 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6061 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6121 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6181 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     6241 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     6301 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     6361 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     6421 ttaatgtgag ttacctcact cattaggcac cccaggcttt acactttatg cttccggctc
     6481 ctatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     6541 attacgccaa gcgcgcaatt a
//
