LOCUS       sequence-469227-        4397 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid used for constructing donor DNA for BXB1-compatible p1 
            integration. You can insert your GOIs into this plasmid by using 
            Golden Gate Assembly..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4397)
  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 4397)
  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: pRW402-P1int-BXB1
FEATURES             Location/Qualifiers
     source          1..4397
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1340..2434
                     /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    2446..2493
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             complement(2662..3477)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2662..3477)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3385..3404
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3469..3488
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3702..4290
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4191..4210
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 actatctatt ttttcagtac ataatttaat taaatcatca tgacctttat ctaattttac
       61 atctcttcta ccaagaccta attctttatt aatactatca aaatctgtta ttctattgcc
      121 ttgaggatct ttgaaacacc aatctgagtc tgaggggttt gtctggtcaa ccaccgcgtg
      181 ctcagtggtg tacggtacaa acctctgatg agacggagag gtcagctttt ctttgctaat
      241 atccagtgaa ctaccaatca ttataggaag ttctctgtca ttgtatatag cacagtaact
      301 taggatttaa gaacctctag aaccagtaga accaggacca gcagtaccat caacagcaga
      361 attagaagct tgagccctag atctcaaacc agatttatac aattcatcca taccaccagt
      421 agaatgtcta ccttcagatc tttcatattg ttcaacaaca gtataatcct cgttatgtga
      481 agtaatgtca agttttctat ctacattata agcacccggc atttgaacag gttttttagc
      541 tttataagta gttttaaaat cagccaaata tctaccacca tctttcaatc tcaaagccat
      601 tttaatatca cctttcaaaa caccatcttc aggatacaat ctttcagtag aagcttccca
      661 acccatagtt tttttttgca taacaggacc atcaggtgga aaattagtac ctctcaattt
      721 aactttataa atcaaagtac catcttccaa agaagtatct tgagtaacag taacagcacc
      781 accatcttca aaattcataa ctctttccca tttaaaacct tcaggaaaag attgtttata
      841 ataatcagga atatcagcag gatgtttaat aaaagctcta gaaccataca taaattgtgg
      901 agacaaaata tcccaagaaa atggcaatgg accaccttta gtaactttca atttagcagt
      961 ttgagtacct tcataaggtc taccttcacc ttcaccttca atttcaaatt catgaccatt
     1021 catagaacct tccatgtgaa ctttaaatct cataaattct ttaataacag cttcaccttt
     1081 agaaaccata tttttatatg tctatgagct tatcacatgt ttctatattt ttctgatatt
     1141 tgaaagttat taatcttttt gtttctatag atcttcctat gtataggtca tccgttatcg
     1201 tctcaagcag tacagaaaat taatttctaa tgtgaacact agtgagatag ctaaagatct
     1261 taattgtgaa aacaatattg aaagtataat aaatacatta aaagaacaaa atagatattt
     1321 tgacaaacaa attgaatata tgtctgcccc taagaagatc gtcgttttgc caggtgacca
     1381 cgttggtcaa gaaatcacag ccgaagccat taaggttctt aaagctattt ctgatgttcg
     1441 ttccaatgtc aagttcgatt tcgaaaatca tttaattggt ggtgctgcta tcgatgctac
     1501 aggtgttcca cttccagatg aggcgctgga agcctccaag aaggctgatg ccgttttgtt
     1561 aggtgctgtg ggtggtccta aatggggaac cggtagtgtt agacctgaac aaggtttact
     1621 aaaaatccgt aaagaacttc aattgtacgc caacttaaga ccatgtaact ttgcatccga
     1681 ctctctttta gacttatctc caatcaagcc acaatttgct aaaggtactg acttcgttgt
     1741 tgtcagagaa ttagtgggag gtatttactt tggtaagaga aaggaggacg atggtgatgg
     1801 tgtcgcttgg gatagtgaac aatacaccgt tccagaagtg caaagaatca caagaatggc
     1861 cgctttcatg gccctacaac atgagccacc attgcctatt tggtccttgg ataaagctaa
     1921 tgttttggcc tcttcaagat tatggagaaa aactgtggag gaaaccatca agaacgaatt
     1981 tcctacattg aaggttcaac atcaattgat tgattctgcc gccatgatcc tagttaagaa
     2041 cccaacccac ctaaatggta ttataatcac cagcaacatg tttggtgata tcatctccga
     2101 tgaagcctcc gttatcccag gttccttggg tttgttgcca tctgcgtcct tggcctcttt
     2161 gccagacaag aacaccgcat ttggtttgta cgaaccatgc cacggttctg ctccagattt
     2221 gccaaagaat aaggtcaacc ctatcgccac tatcttgtct gctgcaatga tgttgaaatt
     2281 gtcattgaac ttgcctgaag aaggtaaggc cattgaagat gcagttaaaa aggttttgga
     2341 tgcaggtatc agaactggtg atttaggtgg ttccaacagt accaccgaag tcggtgatgc
     2401 tgtcgccgaa gaagttaaga aaatccttgc ttaaaagaga gtgagggttt gtaccgtaca
     2461 ccactgagag cgcggtggtt gaccagacaa acctctattt caaagtcgtt aaaagggttg
     2521 ttcaagtcag gagggggaaa cgccaatact ggccctacta ctgctgctgc tgctgctgca
     2581 ccatctagta tcttttaaat taataatgta attcatatat tatagtgcgg ccgcgtgtta
     2641 caaccaatta accaattctg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     2701 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     2761 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     2821 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     2881 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagcttat gcatttcttt
     2941 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     3001 accgttattc attcgtgatt gcgcctgagc gaggcgaaat acgcgatcgc tgttaaaagg
     3061 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     3121 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     3181 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     3241 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     3301 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     3361 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     3421 catgttggaa tttaatcgcg gcctggagca agacgtttcc cgttgaatat ggctcataac
     3481 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgatg atatattttt
     3541 atcttgtgca atgtaacatc agagattttg agacacaacg tggctttgtt gaataaatcg
     3601 aacttttgct gagttgaagg atcagtcatg accaaaatcc cttaacgtga gttttcgttc
     3661 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     3721 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     3781 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     3841 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     3901 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     3961 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4021 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     4081 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     4141 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4201 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4261 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4321 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     4381 gataaccgtg cggccgc
//
