LOCUS       sequence-469026-        8833 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses LaccID evolved variant in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8833)
  AUTHORS   Lee SY, Roh H, Gonzalez-Perez D, Mackey MR, Hoces D, McLaughlin CN, 
            Lin C, Adams SR, Nguyen K, Kim KY, Luginbuhl DJ, Luo L, Udeshi ND, 
            Carr SA, Hernandez-Lopez RA, Ellisman MH, Alcalde M, Ting AY
  TITLE     Directed evolution of LaccID for cell surface proximity labeling and
            electron microscopy.
  JOURNAL   Nat Chem Biol. 2025 Aug 1. doi: 10.1038/s41589-025-01973-6.
  PUBMED    40751001
REFERENCE   2  (bases 1 to 8833)
  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: "Nat Chem 
            Biol. 2025 Aug 1. doi: 10.1038/s41589-025-01973-6."
COMMENT     Sequence Label: pJRoC30-LaccID
FEATURES             Location/Qualifiers
     source          1..8833
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(112..567)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(249..270)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     461..480
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        589..1030
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    589..706
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     1001..1024
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             join(1056..1112,1113..1310,1311..1322)
                     /codon_start=1
                     /gene="MF-alpha-1"
                     /product="N-terminal secretion signal from S. cerevisiae 
                     alpha-factor"
                     /label=alpha-factor secretion signal
                     /note="Cleavage by the Kex2 protease occurs after the 
                     dibasic KR sequence. The EA dipeptides are then removed by 
                     dipeptidyl aminopeptidase A."
                     /translation="MRFPSIFTDVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLE
                     GDSDVAVLPFSNGTNNRLLFINTTIASIAAKEEGVSLEKRGAEA"
     CDS             2832..2855
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2865..2888
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     terminator      2921..3168
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2937..2955)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3245..3262)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3416..4004)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3496..3515)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4175..4966,4967..5035))
                     /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     4798..4817
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(5131..5934)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(5935..6150)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
ORIGIN
        1 tttttatgtt tatctctagt attactcttt agacaaaaaa attgtagtaa gaactattca
       61 tagagtgaat cgaaaacaat acgaaaatgt aaacatttcc tatacgtaga gaaattgtaa
      121 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
      181 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
      241 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
      301 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
      361 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
      421 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
      481 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
      541 cgcttaatgc gccgctacag ggcgcgtggg gatcgatcca ctagtccacg gattagaagc
      601 cgccgagcgg gtgacagccc tccgaaggaa gactctcctc cgtgcgtcct cgtcttcacc
      661 ggtcgcgttc ctgaaacgca gatgtgcctc gcgccgcact gctccgaaca ataaagattc
      721 tacaatacta gcttttatgg ttatgaagag gaaaaattgg cagtaacctg gccccacaaa
      781 ccttcaaatg aacgaatcaa attaacaacc ataggatgat aatgcgatta gttttttagc
      841 cttatttctg gggtaattaa tcagcgaagc gatgattttt gatctattaa cagatatata
      901 aatgcaaaaa ctgcataacc actttaacta atactttcaa cattttcggt ttgtattact
      961 tcttattcaa atgtaataaa agtatcaaca aaaaattgtt aatatacctc tatactttaa
     1021 cgtcaaggag aaaaaactat aggatcatag gatccatgag atttccttca atttttactg
     1081 atgttttatt cgcagcatcc tccgcattag ctgctccagt caacactaca acagaagatg
     1141 aaacggcaca aattccggct gaagctgtca tcggttactc agatttagaa ggggattccg
     1201 atgttgctgt tttgccattt tccaacggca caaataacag gttattgttt ataaatacta
     1261 ctattgccag cattgctgct aaagaagaag gggtatctct cgagaaaaga ggggctgaag
     1321 ctgaattcgc gataggacct gtggcagacc ttcacatcgc caatgcagac atcagcccgg
     1381 atgggttcac tcgttccgct gtgctagcgg gtggaacatt tcccggtccc ttaataacgg
     1441 gtaataaagg agataatttc cggataaacg tcatcaacca gcttacggac acgactatgt
     1501 tgaggtcaac cagtatacat tggcacggat tctttcaaca cgggacaagt tgggctgacg
     1561 gccccgcttt cgtcactcag tgccccatcg caccgggtaa cagtttcctt tacaacttca
     1621 ctgtcccgga tcaagcgggc acgttctggt atcatagtca cctaagtacc cagtattgtg
     1681 acggacttag tggtccgttt gtagtttacg accccaacga tccccatcgt agcttatacg
     1741 atgtggatga tgaaagcact gtaattactc ttgcggactg gtaccacacc ccggctcccc
     1801 agttggccgc tgcgccgccc gtcccagata gcaccctaat aaacgggaaa ggaaggtatg
     1861 ccggaggccc aacgtccccg ttagccgtga taaacgtaga gcagggaaaa agatataggt
     1921 ttcgtcttat atctatttcc tgtgatccga attttacatt tagcattgat ggccataata
     1981 tgactatcat tgaagctgat ggggtgaaca cgcaaccact gaccgtggac tctatccaga
     2041 ttttcgctgg ccaaagatat tccttcgtcc tgaatgcgaa ccagcctgtt ggtaactatt
     2101 ggataagagc gaatccaaat ataggaacga caggattcgc aggtggtatt aatagtgcga
     2161 tcctacgtta tgtaggagct ccagaagcag accctacaac gacccaaaca acatccacaa
     2221 atccagtgaa cgaaacaaac ctacacccct tggagaaccc cggagccccg gggaatccga
     2281 ctccgggtgg tgctgatgtg aacatcaacc ttaacatcgg attctcctca acgggacaat
     2341 tcaccataaa cggtgtttcc ttcataccac ccaccgttcc cgtcttgttg caaatcctgt
     2401 ccggggcgcg tacggcacaa gatctgcttc ctagcggctc tgtttataca ttaccgccga
     2461 acaaagtcat agaaattagt atgccaggcg gcgctagcgc agccgggggg cctcacccgt
     2521 tccatttgca cggccacact tttgatgtgg tacgttcagc gggatcatca acttacaact
     2581 acgtgaatcc agtcagaaga gatgtcgtct ccattggaac ggcgggagat aacgtgacaa
     2641 ttaggttcac tacggacaac ccgggtccat ggttcctaca ttgtcatatc gactggcatc
     2701 ttgaggccgg tttagccgtc gtattcgctg aagacacacc tgatatcgca agcgctaatc
     2761 ccgttccggc tgcgtgggac gacctatgcc ctatttatga tgcgttaagc cccgacgatc
     2821 aagcggccgc tttggaagtt ttgtttcaag gtccaggttc tggtcatcac catcaccatc
     2881 accatcacta atgactcgag catgcatcta gagggccgca tcatgtaatt agttatgtca
     2941 cgcttacatt cacgccctcc ccccacatcc gctctaaccg aaaaggaagg agttagacaa
     3001 cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag aacgttattt
     3061 atatttcaaa tttttctttt ttttctgtac agacgcgtgt acgcatgtaa cattatactg
     3121 aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt taatttgcaa gctgcggccc
     3181 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     3241 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3301 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3361 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3421 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3481 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3541 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3601 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3661 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3721 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3781 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3841 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3901 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3961 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4021 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4081 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     4141 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4201 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4261 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4321 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4381 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4441 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4501 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4561 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4621 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4681 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4741 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4801 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4861 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4921 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4981 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     5041 tcctttttca atgggtaata actgatataa ttaaattgaa gctctaattt gtgagtttag
     5101 tatacatgca tttacttata atacagtttt ttagttttgc tggccgcatc ttctcaaata
     5161 tgcttcccag cctgcttttc tgtaacgttc accctctacc ttagcatccc ttccctttgc
     5221 aaatagtcct cttccaacaa taataatgtc agatcctgta gagaccacat catccacggt
     5281 tctatactgt tgacccaatg cgtctccctt gtcatctaaa cccacaccgg gtgtcataat
     5341 caaccaatcg taaccttcat ctcttccacc catgtctctt tgagcaataa agccgataac
     5401 aaaatctttg tcgctcttcg caatgtcaac agtaccctta gtatattctc cagtagatag
     5461 ggagcccttg catgacaatt ctgctaacat caaaaggcct ctaggttcct ttgttacttc
     5521 ttctgccgcc tgcttcaaac cgctaacaat acctgggccc accacaccgt gtgcattcgt
     5581 aatgtctgcc cattctgcta ttctgtatac acccgcagag tactgcaatt tgactgtatt
     5641 accaatgtca gcaaattttc tgtcttcgaa gagtaaaaaa ttgtacttgg cggataatgc
     5701 ctttagcggc ttaactgtgc cctccatgga aaaatcagtc aagatatcca catgtgtttt
     5761 tagtaaacaa attttgggac ctaatgcttc aactaactcc agtaattcct tggtggtacg
     5821 aacatccaat gaagcacaca agtttgtttg cttttcgtgc atgatattaa atagcttggc
     5881 agcaacagga ctaggatgag tagcagcacg ttccttatat gtagctttcg acatgattta
     5941 tcttcgtttc ctgcaggttt ttgttctgtg cagttgggtt aagaatactg ggcaatttca
     6001 tgtttcttca acactacata tgcgtatata taccaatcta agtctgtgct ccttccttcg
     6061 ttcttccttc tgttcggaga ttaccgaatc aaaaaaattt caaggaaacc gaaatcaaaa
     6121 aaaagaataa aaaaaaaatg atgaattgaa aagctagcag gtacgtagta tatagagaca
     6181 aaatagaaga aaccgttcat aattttctga ccaatgaaga atcatcaacg ctatcacttt
     6241 ctgttcacaa agtatgcgca atccacatcg gtatagaata taatcgggga tgcctttatc
     6301 ttgaaaaaat gcacccgcag cttcgctagt aatcagtaaa cgcgggaagt ggagtcaggc
     6361 tttttttatg gaagagaaaa tagacaccaa agtagccttc ttctaacctt aacggaccta
     6421 cagtgcaaaa agttatcaag agactgcatt atagagcgca caaaggagaa aaaaagtaat
     6481 ctaagatgct ttgttagaaa aatagcgctc tcgggatgca tttttgtaga acaaaaaaga
     6541 agtatagatt ctttgttggt aaaatagcgc tctcgcgttg catttctgtt ctgtaaaaat
     6601 gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt gttttacaaa
     6661 aatgaagcac agattcttcg ttggtaaaat agcgctttcg cgttgcattt ctgttctgta
     6721 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgttct
     6781 acaaaatgaa gcacagatgc ttcgttaaca aagatatgct attgaagtgc aagatggaaa
     6841 cgcagaaaat gaaccgggga tgcgacgtgc aagattacct atgcaataga tgcaatagtt
     6901 tctccaggaa ccgaaataca tacattgtct tccgtaaagc gctagactat atattattat
     6961 acaggttcaa atatactatc tgtttcaggg aaaactccca ggttcggatg ttcaaaattc
     7021 aatgatgggt aacaagtacg atcgtaaatc tgtaaaacag tttgtcggat attaggctgt
     7081 atctcctcaa agcgtattcg aatatcattg agaagctgca gcgtcacatc ggataataat
     7141 gatggcagcc attgtagaag tgccttttgc atttctagtc tctttctcgg tctagctagt
     7201 tttactacat cgcgaagata gaatcttaga tcacactgcc tttgctgagc tggatcaata
     7261 gagtaacaaa agagtggtaa ggcctcgtta aaggacaagg acctgagcgg aagtgtatcg
     7321 tacagtagac ggagtatact agtatagtct atagtccgtg gaattctaag tgccagcttt
     7381 ataatgtcat tctccttact acagacccgc ctgaaagtag acacatcatc atcagtaagc
     7441 tttgacaaaa agcattgagt agctaactct tctatgcaat ctatagctgt tttataaggc
     7501 attcaatgga cagattgagg tttttgaaac atactagtga aattagcctt aatcccttct
     7561 cgaagttaat catgcattat ggtgtaaaaa atgcaactcg cgttgctcta ctttttcccg
     7621 aatttccaaa tacgcagctg gggtgattgc tcgatttcgt aacgaaagtt ttgtttataa
     7681 aaaccgcgaa aaccttctgt aacagataga tttttacagc gctgatatac aatgacatca
     7741 gctgtaatgg aaaataactg aaatatgaat ggcgagagac tgcttgcttg tattaagcaa
     7801 tgtattatgc agcacttcca acctatggtg tacgatgaaa gtaggtgtgt aatcgagacg
     7861 acaaggggga cttttccagt tcctgacaat tataagaaat acaaaacgtt agcatttgca
     7921 tttgttggac atgtactgaa tacagacgac acaccggtaa ttgaaaaaga actggattgg
     7981 cctgatcctg cactagtgta caatacaatt gtcgatcgaa tcataaatca cccagaatta
     8041 tcacagttta tatcggttgc atttattagt cagttaaagg ccaccatcgg agagggttta
     8101 gatattaatg taaaaggcac gctaaaccgc aggggaaagg gtatcagaag gcctaaaggc
     8161 gtatttttta gatacatgga atctccattt gtcaatacaa aggtcactgc attcttctct
     8221 tatcttcgag attataataa aattgcctca gaatatcaca ataatactaa attcattctc
     8281 acgttttcat gtcaagcata ttgggcatct ggcccaaact tctccgcctt gaagaatgtt
     8341 attaggtgct ccataattca tgaatacatt tctaagtttg tggaaagaga acaggataaa
     8401 ggtcatatag gagatcagga gctaccgcct gaagaggacc cttctcgtga actaaacaat
     8461 gtacaacatg aagtcaatag tttaacggaa caagatgcgg aggcggatga aggattgtgg
     8521 ggtgaaatag attcattatg tgaaaaatgg cagtctgaag cggaagatca aactgaggcg
     8581 gagataatag ccgacaggat aattggaaat agccagagga tggcgaacct caaaattcgt
     8641 cgtacaaagt tcaaaagtgt cttgtatcat atactaaagg aactaattca atctcaggga
     8701 accgtaaagg tttatcgcgg tagtagtttt tcacacgatt cgataaagat aagcttacat
     8761 tatgaagagc agcatattac agccgtatgg gtctacttga cagtaaaatt tgaagagcat
     8821 tggaagcctg ttg
//
