LOCUS       sequence-224375-        9489 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of proteins with C-terminal Spot-Tag in S. 
            cerevisiae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9489)
  TITLE     ChromoTek Spot-tag plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9489)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pSpot8
FEATURES             Location/Qualifiers
     source          1..9489
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        65..573
                     /gene="S. cerevisiae TPI1"
                     /label=TPI1 promoter
                     /note="strong constitutive promoter for yeast triose 
                     phosphate isomerase"
     CDS             648..683
                     /codon_start=1
                     /product="engineered Spot-Tag(R) peptide that binds with 
                     high affinity to a nanobody"
                     /label=Spot-Tag
                     /translation="PDRVRAVSHWSS"
     primer_bind     complement(797..816)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(798..816)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(823..840)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(823..839)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(832..854)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      980..1435
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1067..1086)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1277..1298
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1736..2143
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2144..3238
                     /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(3732..3751)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3851..3873
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3979..3995
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3979..3995
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     rep_origin      4555..5897
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5518..5565)
                     /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)."
     promoter        6186..6290
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6291..6359,6360..7151)
                     /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"
     rep_origin      7322..7910
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7811..7830
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8064..8081
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8198..8219
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8234..8239,8240..8257,8258..8264)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8272..8288
                     /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     8277..8299
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8296..8312
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8296..8312
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        8333..8351
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(8940..9044)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtgtttaaag attacggata tttaacttac ttagaataat gccatttttt tgagttataa
       61 taatcctacg ttagtgtgag cgggatttaa actgtgagga ccttaataca ttcagacact
      121 tctgcggtat caccctactt attcccttcg agattatatc taggaaccca tcaggttggt
      181 ggaagattac ccgttctaag acttttcagc ttcctctatt gatgttacac ctggacaccc
      241 cttttctggc atccagtttt taatcttcag tggcatgtga gattctccga aattaattaa
      301 agcaatcaca caattctctc ggataccacc tcggttgaaa ctgacaggtg gtttgttacg
      361 catgctaatg caaaggagcc tatatacctt tggctcggct gctgtaacag ggaatataaa
      421 gggcagcata atttaggagt ttagtgaact tgcaacattt actattttcc cttcttacgt
      481 aaatattttt ctttttaatt ctaaatcaat ctttttcaat tttttgtttg tattcttttc
      541 ttgcttaaat ctataactac aaaaaacaca tacaggaatt caccatgggg atccctaggt
      601 gggcccaagc tttgatatcc gctcgagccc cgggtgctag cggagctcca gatagagtta
      661 gagctgtttc tcattggtct tcttaactag ctaactgaat aaggaacaat gaacgttttt
      721 cctttctctt gttcctagta ttaatgactg accgatacat cccttttttt ttttgtcttt
      781 gtctagctcc aattcgccct atagtgagtc gtattacaat tcactggccg tcgttttaca
      841 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
      901 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
      961 cagcctgaat ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     1021 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     1081 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     1141 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     1201 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     1261 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     1321 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     1381 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttcctga
     1441 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatcg accggtcgag
     1501 gagaacttct agtatatcta catacctaat attattgcct tattaaaaat ggaatcccaa
     1561 caattacatc aaaatccaca ttctcttcaa aatcaattgt cctgtacttc cttgttcatg
     1621 tgtgttcaaa aacgttatat ttataggata attatactct atttctcaac aagtaattgg
     1681 ttgtttggcc gagcggtcta aggcgcctga ttcaagaaat atcttgaccg cagttaactg
     1741 tgggaatact caggtatcgt aagatgcaag agttcgaatc tcttagcaac cattattttt
     1801 ttcctcaaca taacgagaac acacaggggc gctatcgcac agaatcaaat tcgatgactg
     1861 gaaatttttt gttaatttca gaggtcgcct gacgcatata cctttttcaa ctgaaaaatt
     1921 gggagaaaaa ggaaaggtga gagcgccgga accggctttt catatagaat agagaagcgt
     1981 tcatgactaa atgcttgcat cacaatactt gaagttgaca atattattta aggacctatt
     2041 gttttttcca ataggtggtt agcaatcgtc ttactttcta acttttctta ccttttacat
     2101 ttcagcaata tatatatata tatttcaagg atataccatt ctaatgtctg cccctaagaa
     2161 gatcgtcgtt ttgccaggtg accacgttgg tcaagaaatc acagccgaag ccattaaggt
     2221 tcttaaagct atttctgatg ttcgttccaa tgtcaagttc gatttcgaaa atcatttaat
     2281 tggtggtgct gctatcgatg ctacaggtgt tccacttcca gatgaggcgc tggaagcctc
     2341 caagaaggct gatgccgttt tgttaggtgc tgtgggtggt cctaaatggg gtaccggtag
     2401 tgttagacct gaacaaggtt tactaaaaat ccgtaaagaa cttcaattgt acgccaactt
     2461 aagaccatgt aactttgcat ccgactctct tttagactta tctccaatca agccacaatt
     2521 tgctaaaggt actgacttcg ttgttgtcag agaattagtg ggaggtattt actttggtaa
     2581 gagaaaggaa gacgatggtg atggtgtcgc ttgggatagt gaacaataca ccgttccaga
     2641 agtgcaaaga atcacaagaa tggccgcttt catggcccta caacatgagc caccattgcc
     2701 tatttggtcc ttggataaag ctaatgtttt ggcctcttca agattatgga gaaaaactgt
     2761 ggaggaaacc atcaagaacg agttccctac attgaaggtt caacatcaat tgattgattc
     2821 tgccgccatg atcctagtta agaacccaac ccacctaaat ggtattataa tcaccagcaa
     2881 catgtttggt gacatcatct ccgatgaagc ctccgttatc ccaggttcct tgggtttgtt
     2941 gccatctgcg tccttggcct ctttgccaga caagaacacc gcatttggtt tgtacgaacc
     3001 atgccacggt tctgctccag atttgccaaa gaataaggtc aaccctatcg ccactatctt
     3061 gtctgctgca atgatgttga aattgtcatt gaacttgcct gaagaaggta aggccattga
     3121 agatgcagtt aaaaaggttt tggatgcagg tatcagaact ggtgatttag gtggttccaa
     3181 cagtaccacc gaagtcggtg atgctgtcgc cgaagaagtt aagaaaatcc ttgcttaaaa
     3241 agattctctt tttttatgat atttgtacat aaactttata aatgaaattc ataatagaaa
     3301 cgacacgaaa ttacaaaatg gaatatgttc atagggtaga cgaaactata tacgcaatct
     3361 acatacattt atcaagaagg agaaaaagga ggatgtaaag gaatacaggt aagcaaattg
     3421 atactaatgg ctcaacgtga taaggaaaaa gaattgcact ttaacattaa tattgacaag
     3481 gaggagggca ccacacaaaa agttaggtgt aacagaaaat catgaaacta tgattcctaa
     3541 tttatatatt ggaggatttt ctctaaaaaa aaaaaaatac aacaaataaa aaacactcaa
     3601 tgacctgacc atttgatgga gtttaagtca ataccttctt gaaccatttc ccataatggt
     3661 gaaagttccc tcaagaattt tactctgtca gaaacggcct taacgacgta gtcgatatgg
     3721 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     3781 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     3841 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     3901 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     3961 tcttagacgt gcggccgctc tagaactagt ggatcaattc cacggactat agactatact
     4021 agtatactcc gtctactgta cgatacactt ccgctcaggt ccttgtcctt taacgaggcc
     4081 ttaccactct tttgttactc tattgatcca gctcagcaaa ggcagtgtga tctaagattc
     4141 tatcttcgcg atgtagtaaa actagctaga ccgagaaaga gactagaaat gcaaaaggca
     4201 cttctacaat ggctgccatc attattatcc gatgtgacgc tgcagcttct caatgatatt
     4261 cgaatacgct ttgaggagat acagcctaat atccgacaaa ctgttttaca gatttacgat
     4321 cgtacttgtt acccatcatt gaattttgaa catccgaacc tgggagtttt ccctgaaaca
     4381 gatagtatat ttgaacctgt ataataatat atagtctagc gctttacgga agacaatgta
     4441 tgtatttcgg ttcctggaga aactattgca tctattgcat aggtaatctt gcacgtcgca
     4501 tccccggttc attttctgcg tttccatctt gcacttcaat agcatatctt tgttaacgaa
     4561 gcatctgtgc ttcattttgt agaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca
     4621 aagaatctga gctgcatttt tacagaacag aaatgcaacg cgaaagcgct attttaccaa
     4681 cgaagaatct gtgcttcatt tttgtaaaac aaaaatgcaa cgcgagagcg ctaatttttc
     4741 aaacaaagaa tctgagctgc atttttacag aacagaaatg caacgcgaga gcgctatttt
     4801 accaacaaag aatctatact tcttttttgt tctacaaaaa tgcatcccga gagcgctatt
     4861 tttctaacaa agcatcttag attacttttt ttctcctttg tgcgctctat aatgcagtct
     4921 cttgataact ttttgcactg taggtccgtt aaggttagaa gaaggctact ttggtgtcta
     4981 ttttctcttc cataaaaaaa gcctgactcc acttcccgcg tttactgatt actagcgaag
     5041 ctgcgggtgc attttttcaa gataaaggca tccccgatta tattctatac cgatgtggat
     5101 tgcgcatact ttgtgaacag aaagtgatag cgttgatgat tcttcattgg tcagaaaatt
     5161 atgaacggtt tcttctattt tgtctctata tactacgtat aggaaatgtt tacattttcg
     5221 tattgttttc gattcactct atgaatagtt cttactacaa tttttttgtc taaagagtaa
     5281 tactagagat aaacataaaa aatgtagagg tcgagtttag atgcaagttc aaggagcgaa
     5341 aggtggatgg gtaggttata tagggatata gcacagagat atatagcaaa gagatacttt
     5401 tgagcaatgt ttgtggaagc ggtattcgca atattttagt agctcgttac agtccggtgc
     5461 gtttttggtt ttttgaaagt gcgtcttcag agcgcttttg gttttcaaaa gcgctctgaa
     5521 gttcctatac tttctagaga ataggaactt cggaatagga acttcaaagc gtttccgaaa
     5581 acgagcgctt ccgaaaatgc aacgcgagct gcgcacatac agctcactgt tcacgtcgca
     5641 cctatatctg cgtgttgcct gtatatatat atacatgaga agaacggcat agtgcgtgtt
     5701 tatgcttaaa tgcgtactta tatgcgtcta tttatgtagg atgaaaggta gtctagtacc
     5761 tcctgtgata ttatcccatt ccatgcgggg tatcgtatgc ttccttcagc actacccttt
     5821 agctgttcta tatgctgcca ctcctcaatt ggattagtct catccttcaa tgctatcatt
     5881 tcctttgata ttggatcata tgcatagtac cgagaaacta gtgcgaagta gtgatcaggt
     5941 attgctgtta tctgatgagt atacgttgtc ctggccacgg cagaagcacg cttatcgctc
     6001 caatttccca caacattagt caactccgtt aggcccttca ttgaaagaaa tgaggtcatc
     6061 aaatgtcttc caatgtgaga ttttgggcca ttttttatag caaagattga ataaggcgca
     6121 tttttcttca aagctgcggc cgcactctca ctagtacgtc aggtggcact tttcggggaa
     6181 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     6241 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     6301 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     6361 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     6421 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     6481 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     6541 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     6601 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     6661 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     6721 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     6781 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     6841 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     6901 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6961 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     7021 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     7081 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     7141 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     7201 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     7261 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     7321 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     7381 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     7441 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     7501 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     7561 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     7621 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     7681 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     7741 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     7801 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     7861 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     7921 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     7981 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     8041 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     8101 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     8161 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     8221 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     8281 gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc gaaattaacc
     8341 ctcactaaag ggaacaaaag ctggtaccgg gccggccgtc gggccgtcga gcttgatggc
     8401 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     8461 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     8521 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     8581 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     8641 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg
     8701 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     8761 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     8821 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     8881 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     8941 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     9001 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     9061 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     9121 atcacgaggc cctttcgtct tcaagaattg gggatctacg tatggtcatt tcttcttcag
     9181 attccctcat ggagaaagtg cggcagatgt atatgacaga gtcgccagtt tccaagagac
     9241 tttattcagg cacttccatg ataggcaaga gagaagaccc agagatgttg ttgtcctagt
     9301 tacacatggt atttattcca gagtattcct gatgaaatgg tttagatgga catacgaaga
     9361 gtttgaatcg tttaccaatg ttcctaacgg gagcgtaatg gtgatggaac tggacgaatc
     9421 catcaataga tacgtcctga ggaccgtgct acccaaatgg actgattgtg agggagacct
     9481 aactacata
//
