LOCUS       sequence-104903-        6280 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCACS backbone (Amp), LEU2, contains an RFP flanked by BsaI sites 
            with CAGT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6280)
  AUTHORS   Agmon N, Mitchell LA, Cai Y, Ikushima S, Chuang J, Zheng A, Choi WJ,
            Martin JA, Caravelli K, Stracquadanio G, Boeke JD
  TITLE     Yeast Golden Gate (yGG) for the Efficient Assembly of S. cerevisiae 
            Transcription Units.
  JOURNAL   ACS Synth Biol. 2015 Jul 17;4(7):853-9. doi: 10.1021/sb500372z. Epub
            2015 Mar 23.
  PUBMED    25756291
REFERENCE   2  (bases 1 to 6280)
  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/sb500372z";
            journalName: "ACS Synth Biol"; date: "2015-07-17- 17"; volume: "4"; 
            issue: "7"; pages: "853-9"
COMMENT     Sequence Label: pAV10.K5.loxP
FEATURES             Location/Qualifiers
     source          1..6280
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(61..78)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(232..820)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(312..331)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1003..1097)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             join(1115..1183,1184..1975)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1333..1352)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     2146..2164
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(2168..2211)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     promoter        2884..3291
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             3292..4386
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     protein_bind    4686..4719
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(4773..5450)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             5457..5468
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    5486..5502
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5510..5516,5517..5534,5535..5540))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5555..5576
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    5688..5721
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     terminator      join(6260..6280,1..37)
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
ORIGIN
        1 gtcaccaccc tgcccttttt ctttaaaacc gaaaagatta cttcgcgtta tgcaggcttc
       61 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
      121 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
      181 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccacag
      241 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
      301 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
      361 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
      421 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
      481 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
      541 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
      601 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
      661 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
      721 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
      781 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
      841 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
      901 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
      961 aagtatatat gagtaaactt ggtctgacag ctcgaggctt ggattctcac caataaaaaa
     1021 cgcccggcgg caaccgagcg ttctgaacaa atccagatgg agttctgagg tcattactgg
     1081 atctatcaac aggagtccaa gcgagctcga tatcatgagt attcaacatt tccgtgtcgc
     1141 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     1201 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     1261 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     1321 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     1381 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     1441 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     1501 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     1561 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     1621 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     1681 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     1741 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     1801 tgctgataaa tctggagccg gtgagcgtgg atctcgcggt atcattgcag cactggggcc
     1861 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     1921 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaatttct
     1981 ccattttagc ttccttagct cctgaaaatc tcgataactc aaaaaatacg cccggtagtg
     2041 atcttatttc attatggtga aagttggaac ctcttacgtg cccgatcaac tcgagtgcca
     2101 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     2161 aggcagaatt tcagataaaa aaaatcctta gctttcgcta aggatgattt ctggaattcg
     2221 cggccgcgta tccttgcgtt ctctaatcct ttcgtcgtgt atattgtaaa aactgctgaa
     2281 cgagtattga taaaagatat cttggccact aaggggcaga tccccttctg gtgtgataga
     2341 caaccccagg agcatagata acaccaactt gtggtggagg gtcatcgaat tggaattgtc
     2401 tgttggcaac agtagaacag atgctgccct tgctatctgt caaaatgccg ctcttcaaag
     2461 ttactttcaa agcgctgtca ctgctacgac agcttttttt ttagaaacag cagcaatgcc
     2521 ttgacatgta acgtaagaaa agaaaaaaga gatggcagaa gaaatactaa gcgataacgg
     2581 caatgtagag gtgctttttt tatcggaata aatagagaag tcagtaacag tgattgctgt
     2641 ggctccctct ttaatcgtat cggatccaca tacctaatat tattgcctta ttaaaaatgg
     2701 aatcccaaca attacatcaa aatccacatt ctcttcaaaa tcaattgtcc tgtacttcct
     2761 tgttcatgtg tgttcaaaaa cgttatattt ataggataat tatactctat ttctcaacaa
     2821 gtaattggtt gtttggccga gcggtctaag gcgcctgatt caagaaatat cttgaccgca
     2881 gttaactgtg ggaatactca ggtatcgtaa gatgcaagag ttcgaatctc ttagcaacca
     2941 ttattttttt cctcaacata acgagaacac acaggggcgc tatcgcacag aatcaaattc
     3001 gatgactgga aattttttgt taatttcaga ggtcgcctga cgcatatacc tttttcaact
     3061 gaaaaattgg gagaaaaagg aaaggtgaga gcgccggaac cggcttttca tatagaatag
     3121 agaagcgttc atgactaaat gcttgcatca caatacttga agttgacaat attatttaag
     3181 gacctattgt tttttccaat aggtggttag caatcgtctt actttctaac ttttcttacc
     3241 ttttacattt cagcaatata tatatatata tttcaaggat ataccattct aatgtctgcc
     3301 cctaagaaga tcgtcgtttt gccaggtgac cacgttggtc aagaaatcac agccgaagcc
     3361 attaaggttc ttaaagctat ttctgatgtt cgttccaatg tcaagttcga tttcgaaaat
     3421 catttaattg gtggtgctgc tatcgatgct acaggtgttc cacttccaga tgaggcgctg
     3481 gaagcctcca agaaggctga tgccgttttg ttaggtgctg tgggtggtcc taaatggggt
     3541 accggtagtg ttagacctga acaaggttta ctaaaaatcc gtaaagaact tcaattgtac
     3601 gccaacttaa gaccatgtaa ctttgcatcc gactctcttt tagacttatc tccaatcaag
     3661 ccacaatttg ctaaaggtac tgacttcgtt gttgtcagag aattagtggg aggtatttac
     3721 tttggtaaga gaaaggaaga cgatggtgat ggtgtcgctt gggatagtga acaatacacc
     3781 gttccagaag tgcaaagaat cacaagaatg gccgctttca tggccctaca acatgagcca
     3841 ccattgccta tttggtcctt ggataaagct aatgttttgg cctcttcaag attatggaga
     3901 aaaactgtgg aggaaaccat caagaacgaa ttccctacat tgaaggttca acatcaattg
     3961 attgattctg ccgccatgat cctagttaag aacccaaccc acctaaatgg tattataatc
     4021 accagcaaca tgtttggtga tatcatctcc gatgaagcct ccgttatccc aggttccttg
     4081 ggtttgttgc catctgcgtc cttggcctct ttgccagaca agaacaccgc atttggtttg
     4141 tacgaaccat gccacggttc tgctccagat ttgccaaaga ataaggtcaa ccctatcgcc
     4201 actatcttgt ctgctgcaat gatgttgaaa ttgtcattga acttgcctga agaaggtaag
     4261 gccattgaag atgcagttaa aaaggttttg gatgcaggta tcagaactgg tgatttaggt
     4321 ggttccaaca gtaccaccga agtcggtgat gctgtcgccg aagaagttaa gaaaatcctt
     4381 gcttaaaaag attctctttt tttatgatat ttgtacataa actttataaa tgaaattcat
     4441 aatagaaacg acacgaaatt acaaaatgga atatgttcat agggtagacg aaactatata
     4501 cgcaatctac atacatttat caagaaggag aaaaaggagg atgtaaagga atacaggtaa
     4561 gcaaattgat actaatggct caacgtgata aggaaaaaga attgcacttt aacattaata
     4621 ttgacaagga ggagggcacc acacaaaaag ttaggtgtaa cagaaaatca tgaaactagc
     4681 tagcaataac ttcgtataat gtatgctata cgaagttatc agtagagacc gcctggctct
     4741 agtagcgatc tacactagca ctatcagcgt tattaagcac cggtggagtg acgaccttca
     4801 gcacgttcgt actgttcaac gatggtgtag tcttcgttgt gggaggtgat gtccagtttg
     4861 atgtcggttt tgtaagcacc cggcagctga accggttttt tagccatgta ggtggtttta
     4921 acttcagcgt cgtagtgacc accgtctttc agtttcagac gcattttgat ttcacctttc
     4981 agagcaccgt cttccgggta catacgttcg gtggaagctt cccaacccat ggtttttttc
     5041 tgcataaccg gaccgtcgga cgggaagttg gtaccacgca gtttaacttt gtagatgaac
     5101 tcaccgtctt gcagggagga gtcctgggta acggtaacaa caccaccgtc ttcgaagttc
     5161 ataacacgtt cccatttgaa accttccggg aaggacagtt tcaggtagtc cgggatgtca
     5221 gccgggtgtt taacgtaagc tttggaaccg tactggaact gcggggacag gatgtcccaa
     5281 gcgaacggca gcggaccacc tttggtaact ttcagtttag cggtctgggt accttcgtac
     5341 ggacgacctt caccttcacc ttcgatttcg aactcgtgac cgttaacgga accttccata
     5401 cgaactttga aacgcatgaa ctctttgata acgtcttcgg aggaagccat ctagtatttc
     5461 tcctctttct ctagtatgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     5521 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     5581 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5641 tcggccaacg cgcggggaga ggcggtttgc gtattgggtc tcattttata acttcgtata
     5701 atgtatgcta tacgaagtta tactagtgca tgctttggac agtgataccg aagaaggaag
     5761 taaattcagt agcgccgcat gaggtagtag cagaaggagt taaggttgac catggctcac
     5821 ttggcatata ctgttgggca gaaacagtat agtgaaactg ttatcaaacc aatatatttg
     5881 ggctacatgg ctttcctttc ccgaaaaaga caccatttag taattatgtt gtacttttac
     5941 tctctgaact tctgtgaata taaaaaatat actaaataaa aggatgcaac ttccttaaag
     6001 agggcaagat agcgacataa agtggaaaag tgttagcaac attgcgcagg catcgattaa
     6061 tatatgcata tatacttttt acgtagtcta cgttaaattg tcaatagatc aagcagaatc
     6121 ggtagctctg ttaagcgatt aagcccagtg gctttttgtc gcaccgttta ggttcgcgtt
     6181 agcaagatgg aaaaaaaaga acgcgaatca tgtattgacg tatgacgtat taggcagatg
     6241 ggatacgcgg ccgctgcagt ccggcaaaaa agggcaaggt
//
