LOCUS       sequence-471538-        5752 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  2µ plasmid for constitutive expression of BXB1 under RNR2 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5752)
  AUTHORS   Pisera A, Yu Y, Williams RL, Liu CC
  TITLE     Ultra-Efficient Integration of Gene Libraries onto Yeast Cytosolic 
            Plasmids.
  JOURNAL   bioRxiv [Preprint]. 2024 Nov 30:2024.11.29.626108. doi: 
            10.1101/2024.11.29.626108.
  PUBMED    39651169
REFERENCE   2  (bases 1 to 5752)
  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"; journalName: "bioRxiv 
            [Preprint]. 2024 Nov 30:2024.11.29.626108. doi: 
            10.1101/2024.11.29.626108."
COMMENT     Sequence Label: pRW404-2µ-BXB1
FEATURES             Location/Qualifiers
     source          1..5752
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(13..601)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(93..112)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     rep_origin      623..1605
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(1586..1633)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     gene            1852..3208
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        1852..2195
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2196..3005
                     /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(2263..2282)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      3011..3208
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             3971..5476
                     /codon_start=1
                     /transl_table=11
                     /gene="Bxb1"
                     /product="Bxb1 integrase"
                     /label=Bxb1 integrase
                     /note="Bxb1 large serine integrase from Mycobacterium 
                     bacteriophage Bxb1 (Ghosh et al. 2003)."
                     /protein_id="AFF61396.1"
                     /translation="MVRALVVIRLSRVTDATTSPERQLESCQQLCAQRGWDVVGVAEDL
                     DVSGAVDPFDRKRRPNLARWLAFEEQPFDVIVAYRVDRLTRSIRHLQQLVHWAEDHKKL
                     VVSATEAHFDTTTPFAAVVIALMGTVAQMELEAIKERNRSAAHFNIRAGKYRGSLPPWG
                     YLPTRVDGEWRLVPDPVQRERILEVYHRVVDNHEPLHLVAHDLNRRGVLSPKDYFAQLQ
                     GREPQGREWSATALKRSMISGAMLGYATLNGKTVRDDDGAPLVRAEPILTREQLEALRA
                     ELVKTSRAKPAVSTPSLLLRVLFCAVCGEPAYKFAGGGRKHPRYRCRSMGFPKHCGNGT
                     VAMAEWDAFCEEQVLDLLGDAERLEKVWVAGSDSAVELAEVNAELVDLTSLIGSPAYRA
                     GSPQREALDARIAALAARQEELEGLEARPSGWEWRETGQRFGDWWREQDTAAKNTWLRS
                     MNVRLTFDVRGGLTRTIDFGDLQEYEQHLRLGSVVERLHTGMS"
     terminator      5486..5673
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(5541..5565)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
ORIGIN
        1 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
       61 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
      121 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
      181 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
      241 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
      301 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
      361 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
      421 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
      481 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
      541 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
      601 agaagatcct ttgatctttt ctaccgaagc atctgtgctt cattttgtag aacaaaaatg
      661 caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc tgcattttta cagaacagaa
      721 atgcaacgcg aaagcgctat tttaccaacg aagaatctgt gcttcatttt tgtaaaacaa
      781 aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat ttttacagaa
      841 cagaaatgca acgcgagagc gctattttac caacaaagaa tctatacttc ttttttgttc
      901 tacaaaaatg catcccgaga gcgctatttt tctaacaaag catcttagat tacttttttt
      961 ctcctttgtg cgctctataa tgcagtctct tgataacttt ttgcactgta ggtccgttaa
     1021 ggttagaaga aggctacttt ggtgtctatt ttctcttcca taaaaaaagc ctgactccac
     1081 ttcccgcgtt tactgattac tagcgaagct gcgggtgcat tttttcaaga taaaggcatc
     1141 cccgattata ttctataccg atgtggattg cgcatacttt gtgaacagaa agtgatagcg
     1201 ttgatgattc ttcattggtc agaaaattat gaacggtttc ttctattttg tctctatata
     1261 ctacgtatag gaaatgttta cattttcgta ttgttttcga ttcactctat gaatagttct
     1321 tactacaatt tttttgtcta aagagtaata ctagagataa acataaaaaa tgtagaggtc
     1381 gagtttagat gcaagttcaa ggagcgaaag gtggatgggt aggttatata gggatatagc
     1441 acagagatat atagcaaaga gatacttttg agcaatgttt gtggaagcgg tattcgcaat
     1501 attttagtag cccgttacag tccggtgcgt ttttggtttt ttgaaagtgc gtcttcagag
     1561 cgcttttggt tttcaaaagc gctctgaagt tcctatactt tctagagaat aggaacttcg
     1621 gaataggaac ttcaaagcgt ttccgaaaac gagcgcttcc gaaaatgcaa cgcgagctgc
     1681 gcacatacag ctcactgttc acgtcgcacc tatatctgcg tgttgcctgt atatatatat
     1741 acatgagaag aacggcatag tgcgtgttta tgcttaaatg cgtatatgtg ttatgtatag
     1801 gtctagagat ctgtttagct tgcctcgtcc ccgccgggtc acccggccag cgacatggag
     1861 gcccagaata ccctccttga cagtcttgac gtgcgcagct caggggcatg atgtgactgt
     1921 cgcccgtaca tttagcccat acatccccat gtataatcat ttgcatccat acattttgat
     1981 ggccgcacgg cgcgaagcaa aaattacggc tcctcgctgc agacctgcga gcagggaaac
     2041 gctcccctca cagacgcgtt gaattgtccc cacgccgcgc ccctgtagag aaatataaaa
     2101 ggttaggatt tgccactgag gttcttcttt catatacttc cttttaaaat cttgctagga
     2161 tacagttctc acatcacatc cgaacataaa caaccatggg taaggaaaag actcacgttt
     2221 cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg
     2281 ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag
     2341 agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca
     2401 gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc
     2461 ctgatgatgc atggttactc accactgcga tccccggcaa aacagcattc caggtattag
     2521 aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt
     2581 tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc
     2641 aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta
     2701 atggctggcc tgttgaacaa gtctggaaag aaatgcataa gcttttgcca ttctcaccgg
     2761 attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat
     2821 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca
     2881 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat
     2941 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt
     3001 tctaatcagt actgacaata aaaagattct tgttttcaag aacttgtcat ttgtatagtt
     3061 tttttatatt gtagttgttc tattttaatc aaatgttagc gtgatttata ttttttttcg
     3121 cctcgacatc atctgcccag atgcgaagtt aagtgcgcag aaagtaatat catgcgtcaa
     3181 tcgtatgtga atgctggtcg ctatactgct gtcgattcga tactaacgcc gccatccagt
     3241 gtcgaaaacg agctctcgag aacccttaat agtcgaacaa gaagcaggca aagtttagag
     3301 cactgcccct ccgcactcaa aaaagaaaaa actaggagga aaataaaatt ctcaaccaca
     3361 caaacacata aacacataca aatacaaata caagcttatt tacttgacat cgcgcgatct
     3421 tccactattc agcgccgtcc gccctctctc gtgttttttg tttacgcgac aactatgcga
     3481 aatccggagc aacgggcaac cgtttgggga aagaccacac ccacgcgcga tcgccatggc
     3541 aacgaggtcg cacacgcccc acacccagac ctccctgcga gcgggcatgg gtacaatgtc
     3601 cccgttgcca cagacaccac ttcgtagcac agcgcagagc gtagcgtgtt gttgctgctg
     3661 acaaaagaaa atttttctta gcaaagcaaa ggaggggaag cacgggcaga tagcaccgta
     3721 ccataccctt ggaaactcga aatgaacgaa gcaggaaatg agagaatgag agttttgtag
     3781 gtatatatag cggtagtgtt tgcgcgttac catcatcttc tggatctatc tattgttctt
     3841 ttcctcatca ctttcccctt tttcgctctt cttcttgtct tttatttctt tctttttttt
     3901 aattgttccc tcgattggct atctaccaaa gaatccaaac ttaatacacg tatttatttg
     3961 tccaattacc atggtgagag ccctggtagt catccgcctg tcccgcgtca ccgatgctac
     4021 gacttcaccg gagcgtcagc tggagtcttg ccagcagctc tgcgcccagc gcggctggga
     4081 cgtcgtcggg gtagcggagg atctggacgt ctccggggcg gtcgatccgt tcgaccggaa
     4141 gcgcagaccg aacctggccc ggtggctagc gttcgaggag caaccgtttg acgtgatcgt
     4201 ggcgtaccgg gtagatcggt tgacccgatc gatccggcat cttcagcagc tggtccactg
     4261 ggccgaggac cacaagaagc tggtcgtctc cgcgaccgaa gcgcacttcg atacgacgac
     4321 gccgtttgcg gcggtcgtca tcgcgcttat gggaacggtg gcgcagatgg aattagaagc
     4381 gatcaaagag cggaaccgtt cggctgcgca tttcaatatc cgcgccggga aataccgagg
     4441 atccctgccg ccgtggggat acctgcctac gcgcgtggac ggggagtggc ggctggtgcc
     4501 ggaccctgtg cagcgagagc gcatcctcga ggtgtatcac cgcgtcgtcg acaaccacga
     4561 gccgctgcat ctggtggccc acgacctgaa ccggcgtggt gtcctgtcgc cgaaggacta
     4621 cttcgcgcag ctgcaaggcc gcgagccgca gggccgggag tggtcggcta ccgcgctgaa
     4681 gcgatcgatg atctccgggg cgatgctcgg gtacgcgact ctgaacggta agaccgtccg
     4741 agacgacgac ggagccccgc tggtgcgggc tgagccgatc ctgacccgtg agcagctgga
     4801 ggcgctgcgc gccgagctcg tcaagacctc ccgggcgaag cccgcggtgt ctaccccgtc
     4861 gctgctgctg cgggtgttgt tctgcgcggt gtgcggggag cccgcgtaca agttcgccgg
     4921 gggaggacgt aagcacccgc gctaccgctg ccgctcgatg gggttcccga agcactgcgg
     4981 gaacggcacg gtggcgatgg ccgagtggga cgcgttctgc gaggagcagg tactggatct
     5041 gctcggggac gcggagcgtc tggagaaagt ctgggtagcg ggctcggact ccgcggtcga
     5101 actcgcggag gtgaacgcgg agctggtgga cctgacgtcg ctgatcggct ccccggccta
     5161 ccgggcgggc tctccgcagc gagaagcact ggatgcccgt attgcggcgc tggccgcgcg
     5221 gcaagaggag ctggagggcc tggaggctcg cccgtctggc tgggagtggc gcgaaaccgg
     5281 gcagcggttc ggggactggt ggcgggagca ggacaccgcg gcaaagaaca cctggcttcg
     5341 gtcgatgaac gttcggctga cgttcgacgt ccgcggcggg ctgactcgca cgatcgactt
     5401 cggggatctt caggagtacg agcagcatct caggctcggc agcgtggtcg aacggctaca
     5461 caccgggatg tcgtaataac tcgaggcgaa tttcttatga tttatgattt ttattattaa
     5521 ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg ttttaaaacg
     5581 aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc aggtatagca
     5641 tgaggtcgct cttattgacc acacctctac cggcatgccg agcaaatgcc tgcaaatcgc
     5701 tccccatttc agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gc
//
