LOCUS       sequence-250435-        6063 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration cassette for OrthoRep containing mKate driven by 
            promoter 10B2 with a hardcoded polyA tail.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6063)
  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 6063)
  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-A75-RZ-URA3
FEATURES             Location/Qualifiers
     source          1..6063
                     /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"
     misc_RNA        complement(1139..1190)
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             complement(1278..1976)
                     /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(2147..3241)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSVPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMAL*HEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(3492..3510)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3506..3553
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        3620..3711
                     /gene="bla"
                     /label=AmpR promoter
     terminator      4576..4615
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      4646..5234
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5135..5154
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5388..5405
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5420..5559
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5485..5504)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(5656..5847)
                     /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 ggcaattccc
     1141 tgtttcgtcc tcacggactc atcagaccgg aaaacacatc cggtgacagg tttttttttt
     1201 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
     1261 ttttttcatg aggatcctct gtgccccagt ttgctaggga ggtcgcagta tctggccaca
     1321 gccacctcgt gctgctcgac gtaggtctct ttgtcggcct ccttgattct ttccagtctt
     1381 ctgtccacat agtagacgcc gggcatcttg aggttcttag cgggtttctt ggatctgtat
     1441 gtggtcttca agttgcagat caggtggccc ccgcccacga gcttcagggc catgtcggct
     1501 ctgccttcca ggccgccgtc agcggggtac agggtctcgg tggaggcctc ccagccgagt
     1561 gttttcttct gcatcacagg gccgttggat gggaagttca cccctctgat cttgacgttg
     1621 tagatgaggc agccgtcctg gaggctggtg tcctgggtag cggtcagcac gcccccgtct
     1681 tcgtatgtgg tgactctctc ccatgtgaag ccctcgggga aggactgctt aaagaagtcg
     1741 gggatgccct gggtgtggtt gatgaaggtt ttgctgccgt acatgaagct ggtagccagg
     1801 atgtcgaagg cgaaggggag agggccgccc tcgaccgcct tgattctcat ggtctgggtg
     1861 ccctcgtagg gcttgccttc gccctcggat gtgcacttga agtggtggtt gttcacggtg
     1921 ccctccatgt acagcttcat gtgcatgttc tccttaatca gctcgctcac atgcatttta
     1981 catgtctatg agcttatcac atgtttctac atttttctga tatttgaaag ttattaatct
     2041 ttttgtttct atagatcttc ctatgtatag gtcatcccgc ggcgtttcta ttatgaattt
     2101 catttataaa gtttatgtac aaatatcata aaaaaagaga atctttttaa gcaaggattt
     2161 tcttaacttc ttcggcgaca gcatcaccga cttcggtggt actgttggaa ccacctaaat
     2221 caccagttct gatacctgca tccaaaacct ttttaactgc atcttcaatg gccttacctt
     2281 cttcaggcaa gttcaatgac aatttcaaca tcattgcagc agacaagata gtggcgatag
     2341 ggttgacctt attctttggc aaatctggag cagaaccgtg gcatggttcg tacaaaccaa
     2401 atgcggtgtt cttgtctggc aaagaggcca aggacgcaga tggcaacaaa cccaaggaac
     2461 ctgggataac ggaggcttca tcggagatga tatcaccaaa catgttgctg gtgattataa
     2521 taccatttag gtgggttggg ttcttaacta ggatcatggc ggcagaatca atcaattgat
     2581 gttgaacctt caatgtaggg aattcgttct tgatggtttc ctccacagtt tttctccata
     2641 atcttgaaga ggccaaaaca ttagctttat ccaaggacca aataggcaat ggtggctcat
     2701 gttatagggc catgaaagcg gccattcttg tgattctttg cacttctgga acggtgtatt
     2761 gttcactatc ccaagcgaca ccatcaccat cgtcttcctt tctcttacca aagtaaatac
     2821 ctcccactaa ttctctgaca acaacgaagt cagtaccttt agcaaattgt ggcttgattg
     2881 gagataagtc taaaagagag tcggatgcaa agttacatgg tcttaagttg gcgtacaatt
     2941 gaagttcttt acggattttt agtaaacctt gttcaggtct aacactaccg gtaccccatt
     3001 taggaccacc cacagcacct aacaaaacgg catcagcctt cttggaggct tccagcgcct
     3061 catctggaag tggaacacct gtagcatcga tagcagcacc accaattaaa tgattttcga
     3121 aatcgaactt gacattggaa cgaacatcag aaatagcttt aagaacctta atggcttcgg
     3181 ctgtgatttc ttgaccaacg tggtcacctg gcaaaacgac gatcttctta ggaacagaca
     3241 ttttataatt attataaaaa ctttcatata agagatataa aatttaatat ggaagaaata
     3301 agacaagaaa gatacaacca aatgaaagaa gctctaaata gtgttgaagg ttataaagga
     3361 aaaattgtag cctcagactc agattggtgt ttcaaagatc ctcaaggcaa tagaataaca
     3421 gattttgata gtacttctag aagatccggc tgctaacaaa gcccgaaagg aagctgagtt
     3481 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     3541 gaggggtttt ttgctgaaag gaggaactat atccgactag tagcccgcct aatgagcggg
     3601 ctttttttta attcccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3661 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     3721 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     3781 cacccagaaa cgctcgtgaa agtaaaagac gcagaggacc aattgggggc acgagtggga
     3841 tacatagaac tggacttgaa tagcggtaaa atccttgaga gttttcgccc tgaagagcgt
     3901 tttccaatga tgagcacttt caaagttctg ctatgtggag cagtattatc ccgtgtagat
     3961 gcggggcaag agcaactcgg acgacgaata cactattcgc agaatgactt ggttgaatac
     4021 tccccagtga cagaaaagca ccttacggac ggaatgacgg taagagaatt atgtagtgcc
     4081 gccataacga tgagtgataa cactgcggcg aacttacttc tgacaaccat cggtggaccg
     4141 aaggaattaa ccgctttttt gcacaatatg ggagaccatg taactcgcct tgaccgttgg
     4201 gaaccagaac tgaatgaagc cataccaaac gacgagcgag acaccacaat gcctgcggca
     4261 atggcaacaa cattacgcaa actattaact ggcgaactac ttactctggc ttcacggcaa
     4321 caattaatag actggcttga agcggataaa gttgcaggac cactactgcg ttcggcactt
     4381 cctgctggct ggtttattgc tgataaatct ggggcaggag agcgtggttc acggggtatc
     4441 attgccgcac ttggaccaga tggtaagcct tcccgtatcg tagttatcta cacgacgggt
     4501 agtcaggcaa ctatggacga acgaaataga cagattgctg aaataggggc ttcactgatt
     4561 aagcattggt aaaccgatac aattaaaggc tccttttgga gccttttttt ttggacggac
     4621 cggtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     4681 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     4741 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     4801 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     4861 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     4921 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     4981 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5041 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     5101 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     5161 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     5221 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     5281 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     5341 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     5401 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     5461 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     5521 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     5581 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacg
     5641 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     5701 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     5761 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     5821 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     5881 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     5941 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     6001 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttggc
     6061 acc
//
