LOCUS       sequence-465771-        7693 bp    DNA     circular SYN 15-APR-2025
DEFINITION  expresses LaccID on the yeast surface.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7693)
  TITLE     Directed evolution of LaccID for cell surface proximity labeling and
            EM
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7693)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: Aga2p-TEVcs-LaccID-myc_pCTCON2
FEATURES             Location/Qualifiers
     source          1..7693
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    69..572
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        609..713
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(714..782,783..1574)
                     /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     complement(932..951)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1745..2333
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2234..2253
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2487..2504
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2621..2642
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2657..2662,2663..2680,2681..2687)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2695..2711
                     /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)."
     primer_bind     2700..2722
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2719..2735
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2719..2735
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2756..2774
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2804..3455
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    3008..3125
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     3419..3442
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             3774..3785
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3786..3812
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3813..3854
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3855..3875
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             5445..5474
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(5807..5826)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5808..5826)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5833..5850)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5833..5849)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5842..5864)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5990..6445
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6077..6096)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6287..6308
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6548..7222)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(7223..7503)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(7524..7543)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7643..7665
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa
      121 tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta
      181 aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa
      241 tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag
      301 atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt
      361 ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa
      421 aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta
      481 ttttttcttt aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa
      541 tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg
      601 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
      661 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
      721 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      781 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
      841 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
      901 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
      961 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1021 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1081 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1141 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1201 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     1261 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1321 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     1381 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     1441 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     1501 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     1561 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     1621 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     1681 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1741 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1801 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1861 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     1921 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     1981 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     2041 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2101 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     2161 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     2221 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2281 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     2341 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     2401 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     2461 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     2521 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     2581 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     2641 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     2701 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctcgaaatta
     2761 accctcacta aagggaacaa aagctggtac caattccttg aattttcaaa aattcttact
     2821 ttttttttgg atggacgcaa agaagtttaa taatcatatt acatggcatt accaccatat
     2881 acatatccat atctaatctt acttatatgt tgtggaaatg taaagagccc cattatctta
     2941 gcctaaaaaa accttctctt tggaactttc agtaatacgc ttaactgctc attgctatat
     3001 tgaagtacgg attagaagcc gccgagcggg tgacagccct ccgaaggaag actctcctcc
     3061 gtgcgtcctc gtcttcaccg gtcgcgttcc tgaaacgcag atgtgcctcg cgccgcactg
     3121 ctccgaacaa taaagattct acaatactag cttttatggt tatgaagagg aaaaattggc
     3181 agtaacctgg ccccacaaac cttcaaatga acgaatcaaa ttaacaacca taggatgata
     3241 atgcgattag ttttttagcc ttatttctgg ggtaattaat cagcgaagcg atgatttttg
     3301 atctattaac agatatataa atgcaaaaac tgcataacca ctttaactaa tactttcaac
     3361 attttcggtt tgtattactt cttattcaaa tgtaataaaa gatcaacaaa aaattgttaa
     3421 tatacctcta tactttaacg tcaaggagaa aaaaccccgg atcgaattcc ctacttcata
     3481 cattttcaat taagatgcag ttacttcgct gtttttcaat attttctgtt attgcttcag
     3541 ttttagcaca ggaactgaca actatatgcg agcaaatccc ctcaccaact ttagaatcga
     3601 cgccgtactc tttgtcaacg actactattt tggccaacgg gaaggcaatg caaggagttt
     3661 ttgaatatta caaatcagta acgtttgtca gtaattgcgg ttctcacccc tcaacaacta
     3721 gcaaaggcag ccccataaac acacagtatg tttttaagga caatagctcg acgattgaag
     3781 gtagataccc atacgacgtt ccagactacg ctggcaagcc catccccaac cccctgctgg
     3841 gcctggacag caccgagaac ttgtactttc aatctctgca ggctagtggt ggaggaggct
     3901 ctggtggagg cggtagcgga ggcggagggt cggctagcca tatggcgata ggacctgtgg
     3961 cagaccttca catcgccaat gcagacatca gcccggatgg gttcactcgt tccgctgtgc
     4021 tagcgggtgg aacatttccc ggtcccttaa taacgggtaa taaaggagat aatttccgga
     4081 taaacgtcat caaccagctt acggacacga ctatgttgag gtcaaccagt atacattggc
     4141 acggattctt tcaacacggg acaagttggg ctgacggccc cgctttcgtc actcagtgcc
     4201 ccatcgcacc gggtaacagt tttctttaca acttcactgt cccggatcaa gcgggcacgt
     4261 tctggtatca tagtcaccta agtacccagt attgtgacgg acttagtggt ccgtttgtag
     4321 tttacgaccc caacgatccc catcgtagct tatacgatgt ggatgatgaa agcactgtaa
     4381 ttactcttgc ggactggtac cacaccccgg ctccccagtt ggccgctgcg ccgcccgtcc
     4441 cagatagcac cctaataaac gggaaaggaa ggtatgccgg aggcccaacg tccccgttag
     4501 ccgtgataaa cgtagagcag ggaaaaagat ataggtttcg tcttatatct atttcctgtg
     4561 atccgaattt tacatttagc attgatggcc ataatatgac tatcattgaa gctgatgggg
     4621 tgaacacgca accactgacc gtggactcta tccagatttt cgctggccaa agatattcct
     4681 tcgtcctgaa tgcgaaccag cctgttggta actattggat aagagcgaat ccaaatatag
     4741 gaacgacagg attcgcaggt ggtattaata gtgcgatcct acgttatgta ggagctccag
     4801 aagcagaccc tacaacgacc caaacaacat ccacaaatcc agtgaacgaa acaaacctac
     4861 accccttgga gaaccccgga gccccgggga atccgactcc gggtggtgct gatgtgaaca
     4921 tcaaccttaa catcggattc tcctcaacgg gacaattcac cataaacggt gtttccttca
     4981 taccacccac cgttcccgtc ttgttgcaaa tcctgtccgg ggcgcgtacg gcacaagatc
     5041 tgcttcctag cggctctgtt tatacattac cgccgaacaa agtcatagaa attagtatgc
     5101 caggcggcgc tagcgcagcc ggggggcctc acccgttcca tttgcacggc cacacttttg
     5161 atgtggtacg ttcagcggga tcatcaactt acaactacgt gaatccagtc agaagagatg
     5221 tcgtctccat tggaacggcg ggagataacg tgacaattag gttcactacg gacaacccgg
     5281 gtccatggtt cctacattgt catatcgact ggcatcttga ggccggttta gccgtcgtat
     5341 tcgctgaaga cacacctgat atcgcaagcg ctaatcccgt tccggctgcg tgggacgacc
     5401 tatgccctat ttatgatgcg ttaagccccg acgatcaagg atccgaacaa aagcttattt
     5461 ctgaagagga cttgtaatag ctcgagatct gataacaaca gtgtagatgt aacaaaatcg
     5521 actttgttcc cactgtactt ttagctcgta caaaatacaa tatacttttc atttctccgt
     5581 aaacaacatg ttttcccatg taatatcctt ttctattttt cgttccgtta ccaactttac
     5641 acatacttta tatagctatt cacttctata cactaaaaaa ctaagacaat tttaattttg
     5701 ctgcctgcca tatttcaatt tgttataaat tcctataatt tatcctatta gtagctaaaa
     5761 aaagatgaat gtgaatcgaa tcctaagaga attgagctcc aattcgccct atagtgagtc
     5821 gtattacaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     5881 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     5941 ccgcaccgat cgcctttccc aacagttgcg cagcctgaat ggcgaatgga cgcgccctgt
     6001 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     6061 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     6121 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     6181 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     6241 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     6301 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     6361 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     6421 aacaaaatat taacgcttac aatttcctga tgcggtattt tctccttacg catctgtgcg
     6481 gtatttcaca ccgcatagat cggcaagtgc acaaacaata cttaaataaa tactactcag
     6541 taataaccta tttcttagca tttttgacga aatttgctat tttgttagag tcttttacac
     6601 catttgtctc cacacctccg cttacatcaa caccaataac gccatttaat ctaagcgcat
     6661 caccaacatt ttctggcgtc agtccaccag ctaacataaa atgtaagctt tcggggctct
     6721 cttgccttcc aacccagtca gaaatcgagt tccaatccaa aagttcacct gtcccacctg
     6781 cttctgaatc aaacaaggga ataaacgaat gaggtttctg tgaagctgca ctgagtagta
     6841 tgttgcagtc ttttggaaat acgagtcttt taataactgg caaaccgagg aactcttggt
     6901 attcttgcca cgactcatct ccatgcagtt ggacgatatc aatgccgtaa tcattgacca
     6961 gagccaaaac atcctcctta ggttgattac gaaacacgcc aaccaagtat ttcggagtgc
     7021 ctgaactatt tttatatgct tttacaagac ttgaaatttt ccttgcaata accgggtcaa
     7081 ttgttctctt tctattgggc acacatataa tacccagcaa gtcagcatcg gaatctagag
     7141 cacattctgc ggcctctgtg ctctgcaagc cgcaaacttt caccaatgga ccagaactac
     7201 ctgtgaaatt aataacagac atactccaag ctgcctttgt gtgcttaatc acgtatactc
     7261 acgtgctcaa tagtcaccaa tgccctccct cttggccctc tccttttctt ttttcgaccg
     7321 aattaattct taatcggcaa aaaaagaaaa gctccggatc aagattgtac gtaaggtgac
     7381 aagctatttt tcaataaaga atatcttcca ctactgccat ctggcgtcat aactgcaaag
     7441 tacacatata ttacgatgct gttctattaa atgcttccta tattatatat atagtaatgt
     7501 cgtgatctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     7561 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     7621 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     7681 ccgaaacgcg cga
//
