LOCUS       sequence-104886-        6899 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pRS416 backbone, URA3 replaced by KanMX, 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 6899)
  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 6899)
  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: pAV11K
FEATURES             Location/Qualifiers
     source          1..6899
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..45
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     145..164
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        190..410
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     protein_bind    814..847
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            902..2258
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        902..1245
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1246..2055
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1313..1332)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2061..2258
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    2321..2354
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(2438..2893)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2575..2596)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2787..2806
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3023..3045
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3037..3054
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3038..3054
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3064..3083
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3064..3082
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     3107..3123
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     3108..3124
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(3208..3885)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             3892..3903
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    3921..3937
                     /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(3945..3951,3952..3969,3970..3975))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3990..4011
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4126..4142)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4126..4142)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4179..4199)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4179..4197)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4218..4234)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4218..4234)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    4242..4258
                     /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(4266..4272,4273..4290,4291..4296))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4449..4466)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4620..5208)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4700..4719)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5379..6170,6171..6239))
                     /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     6002..6021
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6240..6344)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    6381..6884
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt gtcacagctt
       61 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
      121 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
      181 ccacagcttt tcaattcaat tcatcatttt ttttttattc ttttttttga tttcggtttc
      241 tttgaaattt ttttgattcg gtaatctccg aacagaagga agaacgaagg aaggagcaca
      301 gacttagatt ggtatatata cgcatatgta gtgttgaaga aacatgaaat tgcccagtat
      361 tcttaaccca actgcacaga acaaaaacct gcaggaaacg aagataaatc atgtcgaaag
      421 ctacatataa ggaacgtgct gctactcatc ctagtcctgt tgctgccaag ctatttaata
      481 tcatgcacga aaagcaaaca aacttgtgtg cttcattgga tgttcgtacc accaaggaat
      541 tactggagtt agttgaagca ttaggtccca aaatttgttt actaaaaaca catgtggata
      601 tcttgactga tttttccatg gagggcacag ttaagccgct aaaggcatta tccgccaagt
      661 acaatttttt actcttcgaa gacagaaaat ttgctgacat tggtaataca gtcaaattgc
      721 agtactctgc gggtgtatac taaactcaca aattagagct tcaatttaat tatatcagtt
      781 attacccctc ctcgagcgtc gacaaccctt aatataactt cgtataatgt atgctatacg
      841 aagttattag gtctagagat ctgtttagct tgcctcgtcc ccgccgggtc acccggccag
      901 cgacatggag gcccagaata ccctccttga cagtcttgac gtgcgcagct caggggcatg
      961 atgtgactgt cgcccgtaca tttagcccat acatccccat gtataatcat ttgcatccat
     1021 acattttgat ggccgcacgg cgcgaagcaa aaattacggc tcctcgctgc agacctgcga
     1081 gcagggaaac gctcccctca cagacgcgtt gaattgtccc cacgccgcgc ccctgtagag
     1141 aaatataaaa ggttaggatt tgccactgag gttcttcttt catatacttc cttttaaaat
     1201 cttgctagga tacagttctc acatcacatc cgaacataaa caaccatggg taaggaaaag
     1261 actcacgttt cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     1321 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc
     1381 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     1441 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     1501 atccgtactc ctgatgatgc atggttactc accactgcga tccccggcaa aacagcattc
     1561 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     1621 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     1681 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     1741 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa gcttttgcca
     1801 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     1861 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     1921 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     1981 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     2041 gatgagtttt tctaatcagt actgacaata aaaagattct tgttttcaag aacttgtcat
     2101 ttgtatagtt tttttatatt gtagttgttc tattttaatc aaatgttagc gtgatttata
     2161 ttttttttcg cctcgacatc atctgcccag atgcgaagtt aagtgcgcag aaagtaatat
     2221 catgcgtcaa tcgtatgtga atgctggtcg ctatactgct gtcgattcga tactaacgcc
     2281 gccatccagt gtcgaaaacg agctctcgag aacccttaat ataacttcgt ataatgtatg
     2341 ctatacgaag ttattaggtg atatcagatc cactagtggc ctatgtatgc ggtgtgaaat
     2401 accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaaacgt taatattttg
     2461 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     2521 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     2581 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     2641 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     2701 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     2761 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     2821 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     2881 ctacagggcg cgtcgcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg
     2941 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta
     3001 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc
     3061 gcgtaatacg actcactata gggcgaattg ggtaccgggc cccccctcga ggtcgacggt
     3121 atcgataagc ttgatatcga attcctgcag cccccagtag agaccgcctg gctctagtag
     3181 cgatctacac tagcactatc agcgttatta agcaccggtg gagtgacgac cttcagcacg
     3241 ttcgtactgt tcaacgatgg tgtagtcttc gttgtgggag gtgatgtcca gtttgatgtc
     3301 ggttttgtaa gcacccggca gctgaaccgg ttttttagcc atgtaggtgg ttttaacttc
     3361 agcgtcgtag tgaccaccgt ctttcagttt cagacgcatt ttgatttcac ctttcagagc
     3421 accgtcttcc gggtacatac gttcggtgga agcttcccaa cccatggttt ttttctgcat
     3481 aaccggaccg tcggacggga agttggtacc acgcagttta actttgtaga tgaactcacc
     3541 gtcttgcagg gaggagtcct gggtaacggt aacaacacca ccgtcttcga agttcataac
     3601 acgttcccat ttgaaacctt ccgggaagga cagtttcagg tagtccggga tgtcagccgg
     3661 gtgtttaacg taagctttgg aaccgtactg gaactgcggg gacaggatgt cccaagcgaa
     3721 cggcagcgga ccacctttgg taactttcag tttagcggtc tgggtacctt cgtacggacg
     3781 accttcacct tcaccttcga tttcgaactc gtgaccgtta acggaacctt ccatacgaac
     3841 tttgaaacgc atgaactctt tgataacgtc ttcggaggaa gccatctagt atttctcctc
     3901 tttctctagt atgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3961 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4021 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4081 caacgcgcgg ggagaggcgg tttgcgtatt gggtctcatt ttggggatcc actagttcta
     4141 gagcggccgc caccgcggtg gagctccagc ttttgttccc tttagtgagg gttaattgcg
     4201 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4261 ccacacaaca taggagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagg
     4321 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4381 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4441 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4501 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4561 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4621 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4681 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4741 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4801 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4861 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4921 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4981 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5041 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5101 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5161 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5221 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5281 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     5341 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     5401 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     5461 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     5521 gatccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     5581 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5641 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5701 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5761 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5821 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5881 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5941 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6001 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6061 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6121 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6181 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     6241 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6301 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     6361 gtgccacctg ggtccttttc atcacgtgct ataaaaataa ttataattta aattttttaa
     6421 tataaatata taaattaaaa atagaaagta aaaaaagaaa ttaaagaaaa aatagttttt
     6481 gttttccgaa gatgtaaaag actctagggg gatcgccaac aaatactacc ttttatcttg
     6541 ctcttcctgc tctcaggtat taatgccgaa ttgtttcatc ttgtctgtgt agaagaccac
     6601 acacgaaaat cctgtgattt tacattttac ttatcgttaa tcgaatgtat atctatttaa
     6661 tctgcttttc ttgtctaata aatatatatg taaagtacgc tttttgttga aattttttaa
     6721 acctttgttt attttttttt cttcattccg taactcttct accttcttta tttactttct
     6781 aaaatccaaa tacaaaacat aaaaataaat aaacacagag taaattccca aattattcca
     6841 tcattaaaag atacgaggcg cgtgtaagtt acaggcaagc gatccgtcct aagaaacca
//
