LOCUS       sequence-254746-        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             262..1356
                     /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"
     CDS             1527..2225
                     /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"
     misc_RNA        2313..2364
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     terminator      2372..2536
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2426..2450)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2627..3430)
                     /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"
     CDS             3719..3910
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    4007..4146
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4062..4081
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4161..4178)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4332..4920)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4412..4431)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(4951..4990)
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     promoter        complement(5855..5946)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(6013..6060)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     join(6056..6063,1..11)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
ORIGIN
        1 ttgctcagcg gtggcagcag ccaactcagc ttcctttcgg gctttgttag cagccggatc
       61 ttctagaagt actatcaaaa tctgttattc tattgccttg aggatctttg aaacaccaat
      121 ctgagtctga ggctacaatt tttcctttat aaccttcaac actatttaga gcttctttca
      181 tttggttgta tctttcttgt cttatttctt ccatattaaa ttttatatct cttatatgaa
      241 agtttttata ataattataa aatgtctgtt cctaagaaga tcgtcgtttt gccaggtgac
      301 cacgttggtc aagaaatcac agccgaagcc attaaggttc ttaaagctat ttctgatgtt
      361 cgttccaatg tcaagttcga tttcgaaaat catttaattg gtggtgctgc tatcgatgct
      421 acaggtgttc cacttccaga tgaggcgctg gaagcctcca agaaggctga tgccgttttg
      481 ttaggtgctg tgggtggtcc taaatggggt accggtagtg ttagacctga acaaggttta
      541 ctaaaaatcc gtaaagaact tcaattgtac gccaacttaa gaccatgtaa ctttgcatcc
      601 gactctcttt tagacttatc tccaatcaag ccacaatttg ctaaaggtac tgacttcgtt
      661 gttgtcagag aattagtggg aggtatttac tttggtaaga gaaaggaaga cgatggtgat
      721 ggtgtcgctt gggatagtga acaatacacc gttccagaag tgcaaagaat cacaagaatg
      781 gccgctttca tggccctata acatgagcca ccattgccta tttggtcctt ggataaagct
      841 aatgttttgg cctcttcaag attatggaga aaaactgtgg aggaaaccat caagaacgaa
      901 ttccctacat tgaaggttca acatcaattg attgattctg ccgccatgat cctagttaag
      961 aacccaaccc acctaaatgg tattataatc accagcaaca tgtttggtga tatcatctcc
     1021 gatgaagcct ccgttatccc aggttccttg ggtttgttgc catctgcgtc cttggcctct
     1081 ttgccagaca agaacaccgc atttggtttg tacgaaccat gccacggttc tgctccagat
     1141 ttgccaaaga ataaggtcaa ccctatcgcc actatcttgt ctgctgcaat gatgttgaaa
     1201 ttgtcattga acttgcctga agaaggtaag gccattgaag atgcagttaa aaaggttttg
     1261 gatgcaggta tcagaactgg tgatttaggt ggttccaaca gtaccaccga agtcggtgat
     1321 gctgtcgccg aagaagttaa gaaaatcctt gcttaaaaag attctctttt tttatgatat
     1381 ttgtacataa actttataaa tgaaattcat aatagaaacg ccgcgggatg acctatacat
     1441 aggaagatct atagaaacaa aaagattaat aactttcaaa tatcagaaaa atgtagaaac
     1501 atgtgataag ctcatagaca tgtaaaatgc atgtgagcga gctgattaag gagaacatgc
     1561 acatgaagct gtacatggag ggcaccgtga acaaccacca cttcaagtgc acatccgagg
     1621 gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggcggtc gagggcggcc
     1681 ctctcccctt cgccttcgac atcctggcta ccagcttcat gtacggcagc aaaaccttca
     1741 tcaaccacac ccagggcatc cccgacttct ttaagcagtc cttccccgag ggcttcacat
     1801 gggagagagt caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc
     1861 tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc ccatccaacg
     1921 gccctgtgat gcagaagaaa acactcggct gggaggcctc caccgagacc ctgtaccccg
     1981 ctgacggcgg cctggaaggc agagccgaca tggccctgaa gctcgtgggc gggggccacc
     2041 tgatctgcaa cttgaagacc acatacagat ccaagaaacc cgctaagaac ctcaagatgc
     2101 ccggcgtcta ctatgtggac agaagactgg aaagaatcaa ggaggccgac aaagagacct
     2161 acgtcgagca gcacgaggtg gctgtggcca gatactgcga cctccctagc aaactggggc
     2221 acagaggatc ctcatgaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     2281 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aacctgtcac cggatgtgtt ttccggtctg
     2341 atgagtccgt gaggacgaaa cagggaattg ccgaatttct tatgatttat gatttttatt
     2401 attaaataag ttataaaaaa aataagtgta tacaaatttt aaagtgactc ttaggtttta
     2461 aaacgaaaat tcttattctt gagtaactct ttcctgtagg tcaggttgct ttctcaggta
     2521 tagcatgagg tcgctcaagc ttgagctcgg gtaataactg atataattaa attgaagctc
     2581 taatttgtga gtttagtata cctgcattta cttataatac agttttttag ttttgctggc
     2641 cgcatcttct caaatatgct tcccagcctg cttttctgta acgttcaccc tctaccttag
     2701 catcccttcc ctttgcaaat agtcctcttc caacaataat aatgtcagat cctgtagaga
     2761 ccacatcatc cacggttcta tactgttgac ccaatgcgtc acccttgtca tctaaaccca
     2821 caccgggtgt cataatcaac caatcgtaac cttcatctct tccacccatg tctctttgag
     2881 caataaagcc gataacaaaa tctttgtcgc tcttcgcaat gtcaacagta cccttagtat
     2941 attctccagt agatagggag cccttgcatg acaattctgc taacatcaaa aggcctctag
     3001 gttcctttgt tacttcttct gccgcctgct tcaaaccgct aacaatacct gggcccacca
     3061 caccgtgtgc attcgtaatg tctgcccatt ctgctattct gtatacaccc gcagaatact
     3121 gcaatttgac tgtattacca atgtcagcaa attttctgtc ttcgaagagt aaaaaattgt
     3181 acttggcgga taatgccttt agcggcttaa ctgtgccctc catggaaaaa tcagtcaaga
     3241 tatccacatg tgtttttagt aaacaaattt tgggacctaa tgcttcaact aactccagta
     3301 attccttggt ggtacgaaca tccaatgaag cacacaagtt tgtttgcttt tcgtgcatga
     3361 tattaaatag cttggcagca acaggactag gatgagtagc agcacgttcc ttatatgtag
     3421 ctttatgcat tttataatta ttataaaaac tttcatatat tttaaattaa taatgtaatt
     3481 catatattat agtactctcg agggtgccaa cacctacatc tgtattaacg aagcgctggc
     3541 attgaccctg agtgattttt ctctggtccc gccgcatcca taccgccagt tgtttaccct
     3601 cacaacgttc cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg agcatcctct
     3661 ctcgtttcat cggtatcatt accccatgaa cagaaatccc ccttacacgg aggcatcagt
     3721 gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac
     3781 gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct
     3841 tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga
     3901 aaacctctga cacatgcagc tcccgcgtca cagcttgtct gtaagcggat gccgggagca
     3961 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc
     4021 agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt
     4081 actgagagtg caccattgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     4141 atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     4201 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     4261 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     4321 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     4381 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     4441 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     4501 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     4561 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     4621 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     4681 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     4741 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     4801 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4861 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4921 gaagatcctt tgatcttttc taccggtccg tccaaaaaaa aaggctccaa aaggagcctt
     4981 taattgtatc ggtttaccaa tgcttaatca gtgaagcccc tatttcagca atctgtctat
     5041 ttcgttcgtc catagttgcc tgactacccg tcgtgtagat aactacgata cgggaaggct
     5101 taccatctgg tccaagtgcg gcaatgatac cccgtgaacc acgctctcct gccccagatt
     5161 tatcagcaat aaaccagcca gcaggaagtg ccgaacgcag tagtggtcct gcaactttat
     5221 ccgcttcaag ccagtctatt aattgttgcc gtgaagccag agtaagtagt tcgccagtta
     5281 atagtttgcg taatgttgtt gccattgccg caggcattgt ggtgtctcgc tcgtcgtttg
     5341 gtatggcttc attcagttct ggttcccaac ggtcaaggcg agttacatgg tctcccatat
     5401 tgtgcaaaaa agcggttaat tccttcggtc caccgatggt tgtcagaagt aagttcgccg
     5461 cagtgttatc actcatcgtt atggcggcac tacataattc tcttaccgtc attccgtccg
     5521 taaggtgctt ttctgtcact ggggagtatt caaccaagtc attctgcgaa tagtgtattc
     5581 gtcgtccgag ttgctcttgc cccgcatcta cacgggataa tactgctcca catagcagaa
     5641 ctttgaaagt gctcatcatt ggaaaacgct cttcagggcg aaaactctca aggattttac
     5701 cgctattcaa gtccagttct atgtatccca ctcgtgcccc caattggtcc tctgcgtctt
     5761 ttactttcac gagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5821 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     5881 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5941 aacaaatagg ggaattaaaa aaaagcccgc tcattaggcg ggctactagt cggatatagt
     6001 tcctcctttc agcaaaaaac ccctcaagac ccgtttagag gccccaaggg gttatgctag
     6061 tta
//
