LOCUS       sequence-471364-        5818 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid used for constructing donor DNA for TP901-compatible p1 
            integration. This plasmid is a sfGFP dropout plasmid where sfGFP can
            be replaced by GOIs using Golden Gate Assembly..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5818)
  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 5818)
  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: pYY62-P1int-TP901-GFPdropout
FEATURES             Location/Qualifiers
     source          1..5818
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     68..86
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(124..146)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     RBS             940..951
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             958..1671
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      1683..1754
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1770..1797
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             1834..1845
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1846..1872
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_RNA        2244..2295
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     terminator      2304..2469
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2359..2383)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2470..3564)
                     /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"
     primer_bind     complement(3923..3940)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4094..4682)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4174..4193)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4853..5644,5645..5713))
                     /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     5476..5495
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5714..5818)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
       61 ctataaaaat aggcgtatca cgaggccctt tcgtctcacg cgtttcggtg atgacggtga
      121 aaacctctga cacatgcagc tcccggagac gagtactata atatatgaat tacattatta
      181 atttaaaaga tactagatgg tgcagcagca gcagcagcag tagtagggcc agtattggcg
      241 tttccccctc ctgacttgaa caaccctttt aacgactttg aaatagatag tgacctcttc
      301 aaagagcttt ctctggaaat acggggctca gaagagtcca cattagatga agccattatg
      361 gattgcgagt ttttatttcg tttattcaaa ttaaggtaac taaaaaactc cttttgatga
      421 cctatacata ggaagatcta tagaaacaaa aagattaata actttcaaat atcagaaaaa
      481 tatagaaaca tgtgataagc tcatagacat ataaaaatat gagattccca tctatcttca
      541 ccgctgtttt gttcgctgct tcttctgctt tggctgctcc agctaacacc accaccgaag
      601 acgaaaccgc tcaaatccca gctgaagctg ttatcgacta ctctgacttg gaaggtgact
      661 tcgacgctgc tgctttgcca ttgtctaact ctaccaacaa cggtttgtct tctaccaaca
      721 ccaccatcgc ttctatcgct gctaaggaag aaggtgttca attggacaag agagaagctg
      781 acgcagagac cgaaagtgaa acgtgatttc atgcgtcatt ttgaacattt tgtaaatctt
      841 atttaataat gtgtgcggca attcacattt aatttatgaa tgttttctta acatcgcggc
      901 aactcaagaa acggcaggtt cggatcttag ctactagaga aagaggagaa atactagatg
      961 cgtaaaggcg aagagctgtt cactggtgtc gtccctattc tggtggaact ggatggtgat
     1021 gtcaacggtc ataagttttc cgtgcgtggc gagggtgaag gtgacgcaac taatggtaaa
     1081 ctgacgctga agttcatctg tactactggt aaactgccgg ttccttggcc gactctggta
     1141 acgacgctga cttatggtgt tcagtgcttt gctcgttatc cggaccatat gaagcagcat
     1201 gacttcttca agtccgccat gccggaaggc tatgtgcagg aacgcacgat ttcctttaag
     1261 gatgacggca cgtacaaaac gcgtgcggaa gtgaaatttg aaggcgatac cctggtaaac
     1321 cgcattgagc tgaaaggcat tgactttaaa gaggacggca atatcctggg ccataagctg
     1381 gaatacaatt ttaacagcca caatgtttac atcaccgccg ataaacaaaa aaatggcatt
     1441 aaagcgaatt ttaaaattcg ccacaacgtg gaggatggca gcgtgcagct ggctgatcac
     1501 taccagcaaa acactccaat cggtgatggt cctgttctgc tgccagacaa tcactatctg
     1561 agcacgcaaa gcgttctgtc taaagatccg aacgagaaac gcgatcatat ggttctgctg
     1621 gagttcgtaa ccgcagcggg catcacgcat ggtatggatg aactgtacaa atgaccaggc
     1681 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     1741 cggtgaacgc tctctactag agtcacactg gctcaccttc gggtgggcct ttctgcgttt
     1801 ataggtctcg gttccaagga caatagctcg acgattgaag gtagataccc atacgacgtt
     1861 ccagactacg ctctgcaggc tagtggtggt ggtggttctg gtggtggtgg ttctggtggt
     1921 ggtggttctg ctagcgacgt ccaggaactg acaactatat gcgagcaaat cccctcacca
     1981 actttagaat cgacgccgta ctctttgtca acgactacta ttttggccaa cgggaaggca
     2041 atgcaaggag tttttgaata ttacaaatca gtaacgtttg tcagtaattg cggttctcac
     2101 ccctcaacaa ctagcaaagg cagccccata aacacacagt atgttttttg aatcctaata
     2161 atggccgcaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     2221 aaaaaaaaaa aaaaaaaaaa aaacctgtca ccggatgtgt tttccggtct gatgagtccg
     2281 tgaggacgaa acaggactag gctgcgaatt tcttatgatt tatgattttt attattaaat
     2341 aagttataaa aaaaataagt gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa
     2401 aattcttatt cttgagtaac tctttcctgt aggtcaggtt gctttctcag gtatagcatg
     2461 aggtcgctct taagcaagga ttttcttaac ttcttcggcg acagcatcac cgacttcggt
     2521 ggtactgttg gaaccaccta aatcaccagt tctgatacct gcatccaaaa cctttttaac
     2581 tgcatcttca atggccttac cttcttcagg caagttcaat gacaatttca acatcattgc
     2641 agcagacaag atagtggcga tagggttgac cttattcttt ggcaaatctg gagcagaacc
     2701 gtggcatggt tcgtacaaac caaatgcggt gttcttgtct ggcaaagagg ccaaggacgc
     2761 agatggcaac aaacccaagg aacctgggat aacggaggct tcatcggaga tgatatcacc
     2821 aaacatgttg ctggtgatta taataccatt taggtgggtt gggttcttaa ctaggatcat
     2881 ggcggcagaa tcaatcaatt gatgttgaac cttcaatgta ggaaattcgt tcttgatggt
     2941 ttcctccaca gtttttctcc ataatcttga agaggccaaa acattagctt tatccaagga
     3001 ccaaataggc aatggtggct catgttgtag ggccatgaaa gcggccattc ttgtgattct
     3061 ttgcacttct ggaacggtgt attgttcact atcccaagcg acaccatcac catcgtcctc
     3121 ctttctctta ccaaagtaaa tacctcccac taattctctg acaacaacga agtcagtacc
     3181 tttagcaaat tgtggcttga ttggagataa gtctaaaaga gagtcggatg caaagttaca
     3241 tggtcttaag ttggcgtaca attgaagttc tttacggatt tttagtaaac cttgttcagg
     3301 tctaacacta ccggttcccc atttaggacc acccacagca cctaacaaaa cggcatcagc
     3361 cttcttggag gcttccagcg cctcatctgg aagtggaaca cctgtagcat cgatagcagc
     3421 accaccaatt aaatgatttt cgaaatcgaa cttgacattg gaacgaacat cagaaatagc
     3481 tttaagaacc ttaatggctt cggctgtgat ttcttgacca acgtggtcac ctggcaaaac
     3541 gacgatcttc ttaggggcag acattttata attattataa aaactttcat ataaaaagga
     3601 gttttttagt taccttaatt gaaataaacg aaataaaaac tcgccctcag actcagattg
     3661 gtgtttcaaa gatcctcaag gcaatagaat aacagatttt gatagtatta ataaagaatt
     3721 aggtcttggt agaagagatg taaaattaga taaaggtcat gatgatttaa ttaaattatg
     3781 tactgaaaaa atagatagta ctattaattg cgttgcgctc actgcccgct ttccagtcgg
     3841 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3901 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3961 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4021 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4081 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4141 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4201 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4261 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4321 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4381 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4441 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4501 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     4561 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4621 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4681 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4741 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4801 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4861 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4921 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4981 caatgatacc gcgtgaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5041 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5101 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5161 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5221 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5281 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5341 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5401 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5461 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5521 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5581 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5641 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5701 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5761 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcg
//
