LOCUS       sequence-250436-        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             73..876
                     /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(967..1131)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     1053..1077
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(1169..1867)
                     /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(2038..3132)
                     /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(3383..3401)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3397..3444
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        3511..3602
                     /gene="bla"
                     /label=AmpR promoter
     terminator      4467..4506
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      4537..5125
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5026..5045
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5279..5296
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5311..5450
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5376..5395)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(5547..5738)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ctcgagagta ctataatata tgaattacat tattaattta aaatatatga aagtttttat
       61 aataattata aaatgcataa agctacatat aaggaacgtg ctgctactca tcctagtcct
      121 gttgctgcca agctatttaa tatcatgcac gaaaagcaaa caaacttgtg tgcttcattg
      181 gatgttcgta ccaccaagga attactggag ttagttgaag cattaggtcc caaaatttgt
      241 ttactaaaaa cacatgtgga tatcttgact gatttttcca tggagggcac agttaagccg
      301 ctaaaggcat tatccgccaa gtacaatttt ttactcttcg aagacagaaa atttgctgac
      361 attggtaata cagtcaaatt gcagtattct gcgggtgtat acagaatagc agaatgggca
      421 gacattacga atgcacacgg tgtggtgggc ccaggtattg ttagcggttt gaagcaggcg
      481 gcagaagaag taacaaagga acctagaggc cttttgatgt tagcagaatt gtcatgcaag
      541 ggctccctat ctactggaga atatactaag ggtactgttg acattgcgaa gagcgacaaa
      601 gattttgtta tcggctttat tgctcaaaga gacatgggtg gaagagatga aggttacgat
      661 tggttgatta tgacacccgg tgtgggttta gatgacaagg gtgacgcatt gggtcaacag
      721 tatagaaccg tggatgatgt ggtctctaca ggatctgaca ttattattgt tggaagagga
      781 ctatttgcaa agggaaggga tgctaaggta gagggtgaac gttacagaaa agcaggctgg
      841 gaagcatatt tgagaagatg cggccagcaa aactaaaaaa ctgtattata agtaaatgca
      901 ggtatactaa actcacaaat tagagcttca atttaattat atcagttatt acccgagctc
      961 aagcttgagc gacctcatgc tatacctgag aaagcaacct gacctacagg aaagagttac
     1021 tcaagaataa gaattttcgt tttaaaacct aagagtcact ttaaaatttg tatacactta
     1081 ttttttttat aacttattta ataataaaaa tcataaatca taagaaattc ggcaattgga
     1141 gacgaacctc cgtctcacat gaggatcctc tgtgccccag tttgctaggg aggtcgcagt
     1201 atctggccac agccacctcg tgctgctcga cgtaggtctc tttgtcggcc tccttgattc
     1261 tttccagtct tctgtccaca tagtagacgc cgggcatctt gaggttctta gcgggtttct
     1321 tggatctgta tgtggtcttc aagttgcaga tcaggtggcc cccgcccacg agcttcaggg
     1381 ccatgtcggc tctgccttcc aggccgccgt cagcggggta cagggtctcg gtggaggcct
     1441 cccagccgag tgttttcttc tgcatcacag ggccgttgga tgggaagttc acccctctga
     1501 tcttgacgtt gtagatgagg cagccgtcct ggaggctggt gtcctgggta gcggtcagca
     1561 cgcccccgtc ttcgtatgtg gtgactctct cccatgtgaa gccctcgggg aaggactgct
     1621 taaagaagtc ggggatgccc tgggtgtggt tgatgaaggt tttgctgccg tacatgaagc
     1681 tggtagccag gatgtcgaag gcgaagggga gagggccgcc ctcgaccgcc ttgattctca
     1741 tggtctgggt gccctcgtag ggcttgcctt cgccctcgga tgtgcacttg aagtggtggt
     1801 tgttcacggt gccctccatg tacagcttca tgtgcatgtt ctccttaatc agctcgctca
     1861 catgcatttt acatgtctat gagcttatca catgtttcta catttttctg atatttgaaa
     1921 gttattaatc tttttgtttc tatagatctt cctatgtata ggtcatcccg cggcgtttct
     1981 attatgaatt tcatttataa agtttatgta caaatatcat aaaaaaagag aatcttttta
     2041 agcaaggatt ttcttaactt cttcggcgac agcatcaccg acttcggtgg tactgttgga
     2101 accacctaaa tcaccagttc tgatacctgc atccaaaacc tttttaactg catcttcaat
     2161 ggccttacct tcttcaggca agttcaatga caatttcaac atcattgcag cagacaagat
     2221 agtggcgata gggttgacct tattctttgg caaatctgga gcagaaccgt ggcatggttc
     2281 gtacaaacca aatgcggtgt tcttgtctgg caaagaggcc aaggacgcag atggcaacaa
     2341 acccaaggaa cctgggataa cggaggcttc atcggagatg atatcaccaa acatgttgct
     2401 ggtgattata ataccattta ggtgggttgg gttcttaact aggatcatgg cggcagaatc
     2461 aatcaattga tgttgaacct tcaatgtagg gaattcgttc ttgatggttt cctccacagt
     2521 ttttctccat aatcttgaag aggccaaaac attagcttta tccaaggacc aaataggcaa
     2581 tggtggctca tgttataggg ccatgaaagc ggccattctt gtgattcttt gcacttctgg
     2641 aacggtgtat tgttcactat cccaagcgac accatcacca tcgtcttcct ttctcttacc
     2701 aaagtaaata cctcccacta attctctgac aacaacgaag tcagtacctt tagcaaattg
     2761 tggcttgatt ggagataagt ctaaaagaga gtcggatgca aagttacatg gtcttaagtt
     2821 ggcgtacaat tgaagttctt tacggatttt tagtaaacct tgttcaggtc taacactacc
     2881 ggtaccccat ttaggaccac ccacagcacc taacaaaacg gcatcagcct tcttggaggc
     2941 ttccagcgcc tcatctggaa gtggaacacc tgtagcatcg atagcagcac caccaattaa
     3001 atgattttcg aaatcgaact tgacattgga acgaacatca gaaatagctt taagaacctt
     3061 aatggcttcg gctgtgattt cttgaccaac gtggtcacct ggcaaaacga cgatcttctt
     3121 aggaacaaac attttataat tattataaaa actttcatat aagagatata aaatttaata
     3181 tggaagaaat aagacaagaa agatacaacc aaatgaaaga agctctaaat agtgttgaag
     3241 gttataaagg aaaaattgta gcctcagact cagattggtg tttcaaagat cctcaaggca
     3301 atagaataac agattttgat agtacttcta gaagatccgg ctgctaacaa agcccgaaag
     3361 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     3421 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccgacta gtagcccgcc
     3481 taatgagcgg gctttttttt aattccccta tttgtttatt tttctaaata cattcaaata
     3541 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     3601 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     3661 ctgtttttgc tcacccagaa acgctcgtga aagtaaaaga cgcagaggac caattggggg
     3721 cacgagtggg atacatagaa ctggacttga atagcggtaa aatccttgag agttttcgcc
     3781 ctgaagagcg ttttccaatg atgagcactt tcaaagttct gctatgtgga gcagtattat
     3841 cccgtgtaga tgcggggcaa gagcaactcg gacgacgaat acactattcg cagaatgact
     3901 tggttgaata ctccccagtg acagaaaagc accttacgga cggaatgacg gtaagagaat
     3961 tatgtagtgc cgccataacg atgagtgata acactgcggc gaacttactt ctgacaacca
     4021 tcggtggacc gaaggaatta accgcttttt tgcacaatat gggagaccat gtaactcgcc
     4081 ttgaccgttg ggaaccagaa ctgaatgaag ccataccaaa cgacgagcga gacaccacaa
     4141 tgcctgcggc aatggcaaca acattacgca aactattaac tggcgaacta cttactctgg
     4201 cttcacggca acaattaata gactggcttg aagcggataa agttgcagga ccactactgc
     4261 gttcggcact tcctgctggc tggtttattg ctgataaatc tggggcagga gagcgtggtt
     4321 cacggggtat cattgccgca cttggaccag atggtaagcc ttcccgtatc gtagttatct
     4381 acacgacggg tagtcaggca actatggacg aacgaaatag acagattgct gaaatagggg
     4441 cttcactgat taagcattgg taaaccgata caattaaagg ctccttttgg agcctttttt
     4501 tttggacgga ccggtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4561 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4621 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4681 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4741 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4801 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4861 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4921 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4981 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     5041 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     5101 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     5161 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     5221 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     5281 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     5341 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag
     5401 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     5461 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     5521 aagctgtgac gcgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     5581 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     5641 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     5701 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     5761 tttctgttca tggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     5821 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     5881 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     5941 taggtgttgg cacc
//
