LOCUS       sequence-254747-        5954 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration cassette for OrthoRep containing mKate driven by 
            promoter 10B2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5954)
  AUTHORS   Zhong Z, Ravikumar A, Liu CC
  TITLE     Tunable Expression Systems for Orthogonal DNA Replication.
  JOURNAL   ACS Synth Biol. 2018 Dec 21;7(12):2930-2934. doi: 
            10.1021/acssynbio.8b00400. Epub 2018 Nov 16.
  PUBMED    30408954
REFERENCE   2  (bases 1 to 5954)
  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: 
            "10.1021/acssynbio.8b00400"; journalName: "ACS Synth Biol"; date: 
            "2018-12-21- 21"; volume: "7"; issue: "12"; pages: "2930-2934"
COMMENT     Sequence Label: FDP-P10B2-URA3
FEATURES             Location/Qualifiers
     source          1..5954
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             219..1022
                     /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="MHKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     terminator      complement(1113..1277)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     1199..1223
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(1315..2013)
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MHVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRI
                     KAVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVL
                     TATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMA
                     LKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVAR
                     YCDLPSKLGHR"
     CDS             complement(2184..3278)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MFVPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMAL*HEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(3529..3547)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3543..3590
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        3657..3748
                     /gene="bla"
                     /label=AmpR promoter
     terminator      4613..4652
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      4683..5271
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5172..5191
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5425..5442
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5457..5596
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5522..5541)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(5693..5884)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
       61 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
      121 ttaatacaga tgtaggtgtt ggcaccctcg agagtactat aatatatgaa ttacattatt
      181 aatttaaaat atatgaaagt ttttataata attataaaat gcataaagct acatataagg
      241 aacgtgctgc tactcatcct agtcctgttg ctgccaagct atttaatatc atgcacgaaa
      301 agcaaacaaa cttgtgtgct tcattggatg ttcgtaccac caaggaatta ctggagttag
      361 ttgaagcatt aggtcccaaa atttgtttac taaaaacaca tgtggatatc ttgactgatt
      421 tttccatgga gggcacagtt aagccgctaa aggcattatc cgccaagtac aattttttac
      481 tcttcgaaga cagaaaattt gctgacattg gtaatacagt caaattgcag tattctgcgg
      541 gtgtatacag aatagcagaa tgggcagaca ttacgaatgc acacggtgtg gtgggcccag
      601 gtattgttag cggtttgaag caggcggcag aagaagtaac aaaggaacct agaggccttt
      661 tgatgttagc agaattgtca tgcaagggct ccctatctac tggagaatat actaagggta
      721 ctgttgacat tgcgaagagc gacaaagatt ttgttatcgg ctttattgct caaagagaca
      781 tgggtggaag agatgaaggt tacgattggt tgattatgac acccggtgtg ggtttagatg
      841 acaagggtga cgcattgggt caacagtata gaaccgtgga tgatgtggtc tctacaggat
      901 ctgacattat tattgttgga agaggactat ttgcaaaggg aagggatgct aaggtagagg
      961 gtgaacgtta cagaaaagca ggctgggaag catatttgag aagatgcggc cagcaaaact
     1021 aaaaaactgt attataagta aatgcaggta tactaaactc acaaattaga gcttcaattt
     1081 aattatatca gttattaccc gagctcaagc ttgagcgacc tcatgctata cctgagaaag
     1141 caacctgacc tacaggaaag agttactcaa gaataagaat tttcgtttta aaacctaaga
     1201 gtcactttaa aatttgtata cacttatttt ttttataact tatttaataa taaaaatcat
     1261 aaatcataag aaattcggca attggagacg aacctccgtc tcacatgagg atcctctgtg
     1321 ccccagtttg ctagggaggt cgcagtatct ggccacagcc acctcgtgct gctcgacgta
     1381 ggtctctttg tcggcctcct tgattctttc cagtcttctg tccacatagt agacgccggg
     1441 catcttgagg ttcttagcgg gtttcttgga tctgtatgtg gtcttcaagt tgcagatcag
     1501 gtggcccccg cccacgagct tcagggccat gtcggctctg ccttccaggc cgccgtcagc
     1561 ggggtacagg gtctcggtgg aggcctccca gccgagtgtt ttcttctgca tcacagggcc
     1621 gttggatggg aagttcaccc ctctgatctt gacgttgtag atgaggcagc cgtcctggag
     1681 gctggtgtcc tgggtagcgg tcagcacgcc cccgtcttcg tatgtggtga ctctctccca
     1741 tgtgaagccc tcggggaagg actgcttaaa gaagtcgggg atgccctggg tgtggttgat
     1801 gaaggttttg ctgccgtaca tgaagctggt agccaggatg tcgaaggcga aggggagagg
     1861 gccgccctcg accgccttga ttctcatggt ctgggtgccc tcgtagggct tgccttcgcc
     1921 ctcggatgtg cacttgaagt ggtggttgtt cacggtgccc tccatgtaca gcttcatgtg
     1981 catgttctcc ttaatcagct cgctcacatg cattttacat gtctatgagc ttatcacatg
     2041 tttctacatt tttctgatat ttgaaagtta ttaatctttt tgtttctata gatcttccta
     2101 tgtataggtc atcccgcggc gtttctatta tgaatttcat ttataaagtt tatgtacaaa
     2161 tatcataaaa aaagagaatc tttttaagca aggattttct taacttcttc ggcgacagca
     2221 tcaccgactt cggtggtact gttggaacca cctaaatcac cagttctgat acctgcatcc
     2281 aaaacctttt taactgcatc ttcaatggcc ttaccttctt caggcaagtt caatgacaat
     2341 ttcaacatca ttgcagcaga caagatagtg gcgatagggt tgaccttatt ctttggcaaa
     2401 tctggagcag aaccgtggca tggttcgtac aaaccaaatg cggtgttctt gtctggcaaa
     2461 gaggccaagg acgcagatgg caacaaaccc aaggaacctg ggataacgga ggcttcatcg
     2521 gagatgatat caccaaacat gttgctggtg attataatac catttaggtg ggttgggttc
     2581 ttaactagga tcatggcggc agaatcaatc aattgatgtt gaaccttcaa tgtagggaat
     2641 tcgttcttga tggtttcctc cacagttttt ctccataatc ttgaagaggc caaaacatta
     2701 gctttatcca aggaccaaat aggcaatggt ggctcatgtt atagggccat gaaagcggcc
     2761 attcttgtga ttctttgcac ttctggaacg gtgtattgtt cactatccca agcgacacca
     2821 tcaccatcgt cttcctttct cttaccaaag taaatacctc ccactaattc tctgacaaca
     2881 acgaagtcag tacctttagc aaattgtggc ttgattggag ataagtctaa aagagagtcg
     2941 gatgcaaagt tacatggtct taagttggcg tacaattgaa gttctttacg gatttttagt
     3001 aaaccttgtt caggtctaac actaccggta ccccatttag gaccacccac agcacctaac
     3061 aaaacggcat cagccttctt ggaggcttcc agcgcctcat ctggaagtgg aacacctgta
     3121 gcatcgatag cagcaccacc aattaaatga ttttcgaaat cgaacttgac attggaacga
     3181 acatcagaaa tagctttaag aaccttaatg gcttcggctg tgatttcttg accaacgtgg
     3241 tcacctggca aaacgacgat cttcttagga acaaacattt tataattatt ataaaaactt
     3301 tcatataaga gatataaaat ttaatatgga agaaataaga caagaaagat acaaccaaat
     3361 gaaagaagct ctaaatagtg ttgaaggtta taaaggaaaa attgtagcct cagactcaga
     3421 ttggtgtttc aaagatcctc aaggcaatag aataacagat tttgatagta cttctagaag
     3481 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     3541 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     3601 gaactatatc cgactagtag cccgcctaat gagcgggctt ttttttaatt cccctatttg
     3661 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     3721 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     3781 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tcgtgaaagt
     3841 aaaagacgca gaggaccaat tgggggcacg agtgggatac atagaactgg acttgaatag
     3901 cggtaaaatc cttgagagtt ttcgccctga agagcgtttt ccaatgatga gcactttcaa
     3961 agttctgcta tgtggagcag tattatcccg tgtagatgcg gggcaagagc aactcggacg
     4021 acgaatacac tattcgcaga atgacttggt tgaatactcc ccagtgacag aaaagcacct
     4081 tacggacgga atgacggtaa gagaattatg tagtgccgcc ataacgatga gtgataacac
     4141 tgcggcgaac ttacttctga caaccatcgg tggaccgaag gaattaaccg cttttttgca
     4201 caatatggga gaccatgtaa ctcgccttga ccgttgggaa ccagaactga atgaagccat
     4261 accaaacgac gagcgagaca ccacaatgcc tgcggcaatg gcaacaacat tacgcaaact
     4321 attaactggc gaactactta ctctggcttc acggcaacaa ttaatagact ggcttgaagc
     4381 ggataaagtt gcaggaccac tactgcgttc ggcacttcct gctggctggt ttattgctga
     4441 taaatctggg gcaggagagc gtggttcacg gggtatcatt gccgcacttg gaccagatgg
     4501 taagccttcc cgtatcgtag ttatctacac gacgggtagt caggcaacta tggacgaacg
     4561 aaatagacag attgctgaaa taggggcttc actgattaag cattggtaaa ccgatacaat
     4621 taaaggctcc ttttggagcc tttttttttg gacggaccgg tagaaaagat caaaggatct
     4681 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4741 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4801 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     4861 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4921 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4981 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     5041 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     5101 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     5161 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     5221 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     5281 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     5341 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     5401 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     5461 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     5521 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     5581 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     5641 tctgctcccg gcatccgctt acagacaagc tgtgacgcgg gagctgcatg tgtcagaggt
     5701 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     5761 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     5821 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     5881 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg gtaatgatac cgatgaaacg
     5941 agagaggatg ctca
//
