LOCUS       sequence-81070-e        5757 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration cassette to create p1-Short-Leu-mKate with p2 ORF10 
            UCS>mKate.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5757)
  AUTHORS   Ravikumar A, Arrieta A, Liu CC
  TITLE     An orthogonal DNA replication system in yeast.
  JOURNAL   Nat Chem Biol. 2014 Mar;10(3):175-7. doi: 10.1038/nchembio.1439. 
            Epub 2014 Feb 2.
  PUBMED    24487693
REFERENCE   2  (bases 1 to 5757)
  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/nchembio.1439"; journalName: "Nat Chem Biol"; date: 
            "2014-03"; volume: "10"; issue: "3"; pages: "175-7"
COMMENT     Sequence Label: pCCL-GKL1RC-Short-LEUg1mKateg2GKL2ORF10UCS
FEATURES             Location/Qualifiers
     source          1..5757
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             912..2006
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MHVPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     terminator      complement(2077..2242)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     2163..2187
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2258..2956)
                     /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"
     primer_bind     complement(3181..3199)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3195..3242
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        3309..3400
                     /gene="bla"
                     /label=AmpR promoter
     terminator      4265..4304
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      4335..4923
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4824..4843
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5077..5094
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5109..5248
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5174..5193)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5334..5356
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5350..5541)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ctcgagagta cttataataa ttttgaagaa agtacatata tagcatggta tacaaatgta
       61 gatatagata taggtttgtc tgaaggtcat aatatagaat atatcccctt tgattcttat
      121 ggaaatatag gttattcttg gtctaaaaaa ggtaaaatat tcgaaaaata tataaaagac
      181 gtgctgtaca aattaaaaat aaagtatgaa aaacaaaaca ataaagttaa aagaaatgtt
      241 atcaaaatta ttatgaacag tttatggggc aaattcgcac aaaaatgggt aaattttgag
      301 tattttataa aatcagaaga tgatatagat tttgagtcag aagaggcata taagatatgg
      361 gacactgatt ttatgctgat aaagaaaatt aaagaatcta cttattcatc taaacctata
      421 caaaatggag tatttacatt aagttgggca agataccaca tgaaaagtat atgggatgca
      481 ggggctaaag aaggagcaga atgtatctat tcggacacag atagtatttt tgtacataaa
      541 gaacatttta aaaagaatgc taaatttatg ttaaatggtt taaaagttcc tattatagga
      601 tcagaagtag gacaattaga attagaatgt gagtttgata aattgttatg tgcaggtaaa
      661 aagcaataca tgggatttta tacttatttt caagatggaa aaccatgtat aaaagaaaag
      721 aaaagattta agggtattcc tagtaattat ataatacctg aattatatgc tcatttactt
      781 tcaggtgcag acaaagaagc taaaatacaa tttttgaaat ttagaagaga atggggatca
      841 gttaaaggat atatagaaaa taagaccgtg aaagctactt aatatatgaa agtttttata
      901 ataattataa aatgcacgtt cctaagaaga tcgtcgtttt gccaggtgac cacgttggtc
      961 aagaaatcac agccgaagcc attaaggttc ttaaagctat ttctgatgtt cgttccaatg
     1021 tcaagttcga tttcgaaaat catttaattg gtggtgctgc tatcgatgct acaggtgttc
     1081 cacttccaga tgaggcgctg gaagcctcca agaaggctga tgccgttttg ttaggtgctg
     1141 tgggtggtcc taaatggggt accggtagtg ttagacctga acaaggttta ctaaaaatcc
     1201 gtaaagaact tcaattgtac gccaacttaa gaccatgtaa ctttgcatcc gactctcttt
     1261 tagacttatc tccaatcaag ccacaatttg ctaaaggtac tgacttcgtt gttgtcagag
     1321 aattagtggg aggtatttac tttggtaaga gaaaggaaga cgatggtgat ggtgtcgctt
     1381 gggatagtga acaatacacc gttccagaag tgcaaagaat cacaagaatg gccgctttca
     1441 tggccctaca acatgagcca ccattgccta tttggtcctt ggataaagct aatgttttgg
     1501 cctcttcaag attatggaga aaaactgtgg aggaaaccat caagaacgaa ttccctacat
     1561 tgaaggttca acatcaattg attgattctg ccgccatgat cctagttaag aacccaaccc
     1621 acctaaatgg tattataatc accagcaaca tgtttggtga tatcatctcc gatgaagcct
     1681 ccgttatccc aggttccttg ggtttgttgc catctgcgtc cttggcctct ttgccagaca
     1741 agaacaccgc atttggtttg tacgaaccat gccacggttc tgctccagat ttgccaaaga
     1801 ataaggtcaa ccctatcgcc actatcttgt ctgctgcaat gatgttgaaa ttgtcattga
     1861 acttgcctga agaaggtaag gccattgaag atgcagttaa aaaggttttg gatgcaggta
     1921 tcagaactgg tgatttaggt ggttccaaca gtaccaccga agtcggtgat gctgtcgccg
     1981 aagaagttaa gaaaatcctt gcttaaaaag attctctttt tttatgatat ttgtacataa
     2041 actttataaa tgaaattcat aatagaaacg gagctcgagc gacctcatgc tatacctgag
     2101 aaagcaacct gacctacagg aaagagttac tcaagaataa gaattttcgt tttaaaacct
     2161 aagagtcact ttaaaatttg tatacactta ttttttttat aacttattta ataataaaaa
     2221 tcataaatca taagaaattc gcctcgagtt aggatcctct gtgccccagt ttgctaggga
     2281 ggtcgcagta tctggccaca gccacctcgt gctgctcgac gtaggtctct ttgtcggcct
     2341 ccttgattct ttccagtctt ctgtccacat agtagacgcc gggcatcttg aggttcttag
     2401 cgggtttctt ggatctgtat gtggtcttca agttgcagat caggtggccc ccgcccacga
     2461 gcttcagggc catgtcggct ctgccttcca ggccgccgtc agcggggtac agggtctcgg
     2521 tggaggcctc ccagccgagt gttttcttct gcatcacagg gccgttggat gggaagttca
     2581 cccctctgat cttgacgttg tagatgaggc agccgtcctg gaggctggtg tcctgggtag
     2641 cggtcagcac gcccccgtct tcgtatgtgg tgactctctc ccatgtgaag ccctcgggga
     2701 aggactgctt aaagaagtcg gggatgccct gggtgtggtt gatgaaggtt ttgctgccgt
     2761 acatgaagct ggtagccagg atgtcgaagg cgaaggggag agggccgccc tcgaccgcct
     2821 tgattctcat ggtctgggtg ccctcgtagg gcttgccttc gccctcggat gtgcacttga
     2881 agtggtggtt gttcacggtg ccctccatgt acagcttcat gtgcatgttc tccttaatca
     2941 gctcgctcac atgcatttta catgtctatg agcttatcat atatttctac atttttctga
     3001 tatttgaaag ttattaatct ttttgtttct atagatcttc ctatgtatag gtcatcacgc
     3061 gtcctcagac tcagattggt gtaaagatcc tcaaggcaat agaataacag attttgatag
     3121 tacttctaga agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     3181 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     3241 tgctgaaagg aggaactata tccgactagt agcccgccta atgagcgggc ttttttttaa
     3301 ttcccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3361 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     3421 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     3481 gctcgtgaaa gtaaaagacg cagaggacca attgggggca cgagtgggat acatagaact
     3541 ggacttgaat agcggtaaaa tccttgagag ttttcgccct gaagagcgtt ttccaatgat
     3601 gagcactttc aaagttctgc tatgtggagc agtattatcc cgtgtagatg cggggcaaga
     3661 gcaactcgga cgacgaatac actattcgca gaatgacttg gttgaatact ccccagtgac
     3721 agaaaagcac cttacggacg gaatgacggt aagagaatta tgtagtgccg ccataacgat
     3781 gagtgataac actgcggcga acttacttct gacaaccatc ggtggaccga aggaattaac
     3841 cgcttttttg cacaatatgg gagaccatgt aactcgcctt gaccgttggg aaccagaact
     3901 gaatgaagcc ataccaaacg acgagcgaga caccacaatg cctgcggcaa tggcaacaac
     3961 attacgcaaa ctattaactg gcgaactact tactctggct tcacggcaac aattaataga
     4021 ctggcttgaa gcggataaag ttgcaggacc actactgcgt tcggcacttc ctgctggctg
     4081 gtttattgct gataaatctg gggcaggaga gcgtggttca cggggtatca ttgccgcact
     4141 tggaccagat ggtaagcctt cccgtatcgt agttatctac acgacgggta gtcaggcaac
     4201 tatggacgaa cgaaatagac agattgctga aataggggct tcactgatta agcattggta
     4261 aaccgataca attaaaggct ccttttggag cctttttttt tggacggacc ggtagaaaag
     4321 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     4381 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4441 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     4501 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     4561 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     4621 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     4681 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     4741 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     4801 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     4861 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     4921 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     4981 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     5041 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     5101 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     5161 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     5221 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     5281 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     5341 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     5401 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     5461 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     5521 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatggggta
     5581 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     5641 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     5701 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tggcacc
//
