LOCUS       sequence-220576-        9481 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 9481)
  TITLE     ChromoTek Spot-tag plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9481)
  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            "Unpublished"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..9481
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             73..108
                     /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(222..241)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(223..241)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(248..265)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(248..264)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(257..279)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      405..860
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(492..511)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     702..723
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1161..1568
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             1569..2663
                     /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
                     LIDSATMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(3157..3176)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3276..3298
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3404..3420
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3404..3420
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     rep_origin      3980..5322
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(4943..4990)
                     /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        5611..5715
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5716..5784,5785..6576)
                     /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      6747..7335
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7236..7255
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7489..7506
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7623..7644
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7659..7664,7665..7682,7683..7689)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7697..7713
                     /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     7702..7724
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7721..7737
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7721..7737
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        7758..7776
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(8357..8461)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        8971..9479
                     /gene="S. cerevisiae TPI1"
                     /label=TPI1 promoter
                     /note="strong constitutive promoter for yeast triose 
                     phosphate isomerase"
ORIGIN
        1 gaattcacca tggggatccc taggtgggcc caagctttga tatccgctcg agccccgggt
       61 gctagcggag ctccagatag agttagagct gtttctcatt ggtcttctta actagctaac
      121 tgaataagga acaatgaacg tttttccttt ctcttgttcc tagtattaat gactgaccga
      181 tacatccctt tttttttttg tctttgtcta gctccaattc gccctatagt gagtcgtatt
      241 acaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
      301 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
      361 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg
      421 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
      481 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
      541 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
      601 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
      661 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
      721 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
      781 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
      841 aatattaacg cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
      901 tcacaccgca tatcgaccgg tcgaggagaa cttctagtat atctacatac ctaatattat
      961 tgccttatta aaaatggaat cccaacaatt acatcaaaat ccacattctc ttcaaaatca
     1021 attgtcctgt acttccttgt tcatgtgtgt tcaaaaacgt tatatttata ggataattat
     1081 actctatttc tcaacaagta attggttgtt tggccgagcg gtctaaggcg cctgattcaa
     1141 gaaatatctt gaccgcagtt aactgtggga atactcaggt atcgtaagat gcaagagttc
     1201 gaatctctta gcaaccatta tttttttcct caacataacg agaacacaca ggggcgctat
     1261 cgcacagaat caaattcgat gactggaaat tttttgttaa tttcagaggt cgcctgacgc
     1321 atataccttt ttcaactgaa aaattgggag aaaaaggaaa ggtgagagcg ccggaaccgg
     1381 cttttcatat agaatagaga agcgttcatg actaaatgct tgcatcacaa tacttgaagt
     1441 tgacaatatt atttaaggac ctattgtttt ttccaatagg tggttagcaa tcgtcttact
     1501 ttctaacttt tcttaccttt tacatttcag caatatatat atatatattt caaggatata
     1561 ccattctaat gtctgcccct aagaagatcg tcgttttgcc aggtgaccac gttggtcaag
     1621 aaatcacagc cgaagccatt aaggttctta aagctatttc tgatgttcgt tccaatgtca
     1681 agttcgattt cgaaaatcat ttaattggtg gtgctgctat cgatgctaca ggtgttccac
     1741 ttccagatga ggcgctggaa gcctccaaga aggctgatgc cgttttgtta ggtgctgtgg
     1801 gtggtcctaa atggggtacc ggtagtgtta gacctgaaca aggtttacta aaaatccgta
     1861 aagaacttca attgtacgcc aacttaagac catgtaactt tgcatccgac tctcttttag
     1921 acttatctcc aatcaagcca caatttgcta aaggtactga cttcgttgtt gtcagagaat
     1981 tagtgggagg tatttacttt ggtaagagaa aggaagacga tggtgatggt gtcgcttggg
     2041 atagtgaaca atacaccgtt ccagaagtgc aaagaatcac aagaatggcc gctttcatgg
     2101 ccctacaaca tgagccacca ttgcctattt ggtccttgga taaagctaat gttttggcct
     2161 cttcaagatt atggagaaaa actgtggagg aaaccatcaa gaacgagttc cctacattga
     2221 aggttcaaca tcaattgatt gattctgcca ccatgatcct agttaagaac ccaacccacc
     2281 taaatggtat tataatcacc agcaacatgt ttggtgacat catctccgat gaagcctccg
     2341 ttatcccagg ttccttgggt ttgttgccat ctgcgtcctt ggcctctttg ccagacaaga
     2401 acaccgcatt tggtttgtac gaaccatgcc acggttctgc tccagatttg ccaaagaata
     2461 aggtcaaccc tatcgccact atcttgtctg ctgcaatgat gttgaaattg tcattgaact
     2521 tgcctgaaga aggtaaggcc attgaagatg cagttaaaaa ggttttggat gcaggtatca
     2581 gaactggtga tttaggtggt tccaacagta ccaccgaagt cggtgatgct gtcgccgaag
     2641 aagttaagaa aatccttgct taaaaagatt ctcttttttt atgatatttg tacataaact
     2701 ttataaatga aattcataat agaaacgaca cgaaattaca aaatggaata tgttcatagg
     2761 gtagacgaaa ctatatacgc aatctacata catttatcaa gaaggagaaa aaggaggatg
     2821 taaaggaata caggtaagca aattgatact aatggctcaa cgtgataagg aaaaagaatt
     2881 gcactttaac attaatattg acaaggagga gggcaccaca caaaaagtta ggtgtaacag
     2941 aaaatcatga aactatgatt cctaatttat atattggagg attttctcta aaaaaaaaaa
     3001 aatacaacaa ataaaaaaca ctcaatgacc tgaccatttg atggagttta agtcaatacc
     3061 ttcttgaacc atttcccata atggtgaaag ttccctcaag aattttactc tgtcagaaac
     3121 ggccttaacg acgtagtcga tatggtgcac tctcagtaca atctgctctg atgccgcata
     3181 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     3241 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     3301 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     3361 ggttaatgtc atgataataa tggtttctta gacgtgcggc cgctctagaa ctagtggatc
     3421 aattccacgg actatagact atactagtat actccgtcta ctgtacgata cacttccgct
     3481 caggtccttg tcctttaacg aggccttacc actcttttgt tactctattg atccagctca
     3541 gcaaaggcag tgtgatctaa gattctatct tcgcgatgta gtaaaactag ctagaccgag
     3601 aaagagacta gaaatgcaaa aggcacttct acaatggctg ccatcattat tatccgatgt
     3661 gacgctgcag cttctcaatg atattcgaat acgctttgag gagatacagc ctaatatccg
     3721 acaaactgtt ttacagattt acgatcgtac ttgttaccca tcattgaatt ttgaacatcc
     3781 gaacctggga gttttccctg aaacagatag tatatttgaa cctgtataat aatatatagt
     3841 ctagcgcttt acggaagaca atgtatgtat ttcggttcct ggagaaacta ttgcatctat
     3901 tgcataggta atcttgcacg tcgcatcccc ggttcatttt ctgcgtttcc atcttgcact
     3961 tcaatagcat atctttgtta acgaagcatc tgtgcttcat tttgtagaac aaaaatgcaa
     4021 cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc atttttacag aacagaaatg
     4081 caacgcgaaa gcgctatttt accaacgaag aatctgtgct tcatttttgt aaaacaaaaa
     4141 tgcaacgcga gagcgctaat ttttcaaaca aagaatctga gctgcatttt tacagaacag
     4201 aaatgcaacg cgagagcgct attttaccaa caaagaatct atacttcttt tttgttctac
     4261 aaaaatgcat cccgagagcg ctatttttct aacaaagcat cttagattac tttttttctc
     4321 ctttgtgcgc tctataatgc agtctcttga taactttttg cactgtaggt ccgttaaggt
     4381 tagaagaagg ctactttggt gtctattttc tcttccataa aaaaagcctg actccacttc
     4441 ccgcgtttac tgattactag cgaagctgcg ggtgcatttt ttcaagataa aggcatcccc
     4501 gattatattc tataccgatg tggattgcgc atactttgtg aacagaaagt gatagcgttg
     4561 atgattcttc attggtcaga aaattatgaa cggtttcttc tattttgtct ctatatacta
     4621 cgtataggaa atgtttacat tttcgtattg ttttcgattc actctatgaa tagttcttac
     4681 tacaattttt ttgtctaaag agtaatacta gagataaaca taaaaaatgt agaggtcgag
     4741 tttagatgca agttcaagga gcgaaaggtg gatgggtagg ttatataggg atatagcaca
     4801 gagatatata gcaaagagat acttttgagc aatgtttgtg gaagcggtat tcgcaatatt
     4861 ttagtagctc gttacagtcc ggtgcgtttt tggttttttg aaagtgcgtc ttcagagcgc
     4921 ttttggtttt caaaagcgct ctgaagttcc tatactttct agagaatagg aacttcggaa
     4981 taggaacttc aaagcgtttc cgaaaacgag cgcttccgaa aatgcaacgc gagctgcgca
     5041 catacagctc actgttcacg tcgcacctat atctgcgtgt tgcctgtata tatatataca
     5101 tgagaagaac ggcatagtgc gtgtttatgc ttaaatgcgt acttatatgc gtctatttat
     5161 gtaggatgaa aggtagtcta gtacctcctg tgatattatc ccattccatg cggggtatcg
     5221 tatgcttcct tcagcactac cctttagctg ttctatatgc tgccactcct caattggatt
     5281 agtctcatcc ttcaatgcta tcatttcctt tgatattgga tcatatgcat agtaccgaga
     5341 aactagtgcg aagtagtgat caggtattgc tgttatctga tgagtatacg ttgtcctggc
     5401 cacggcagaa gcacgcttat cgctccaatt tcccacaaca ttagtcaact ccgttaggcc
     5461 cttcattgaa agaaatgagg tcatcaaatg tcttccaatg tgagattttg ggccattttt
     5521 tatagcaaag attgaataag gcgcattttt cttcaaagct gcggccgcac tctcactagt
     5581 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5641 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     5701 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5761 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5821 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5881 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5941 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     6001 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     6061 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     6121 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     6181 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     6241 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     6301 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     6361 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     6421 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     6481 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     6541 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6601 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6661 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6721 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6781 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6841 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6901 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6961 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     7021 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     7081 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     7141 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     7201 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     7261 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     7321 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     7381 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     7441 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     7501 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     7561 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     7621 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     7681 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     7741 gattacgcca agctcgaaat taaccctcac taaagggaac aaaagctggt accgggccgg
     7801 ccgtcgagct tgatggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     7861 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     7921 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     7981 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     8041 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     8101 gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     8161 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     8221 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     8281 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     8341 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     8401 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     8461 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     8521 cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattgggg atctacgtat
     8581 ggtcatttct tcttcagatt ccctcatgga gaaagtgcgg cagatgtata tgacagagtc
     8641 gccagtttcc aagagacttt attcaggcac ttccatgata ggcaagagag aagacccaga
     8701 gatgttgttg tcctagttac acatggtatt tattccagag tattcctgat gaaatggttt
     8761 agatggacat acgaagagtt tgaatcgttt accaatgttc ctaacgggag cgtaatggtg
     8821 atggaactgg acgaatccat caatagatac gtcctgagga ccgtgctacc caaatggact
     8881 gattgtgagg gagacctaac tacatagtgt ttaaagatta cggatattta acttacttag
     8941 aataatgcca tttttttgag ttataataat cctacgttag tgtgagcggg atttaaactg
     9001 tgaggacctt aatacattca gacacttctg cggtatcacc ctacttattc ccttcgagat
     9061 tatatctagg aacccatcag gttggtggaa gattacccgt tctaagactt ttcagcttcc
     9121 tctattgatg ttacacctgg acaccccttt tctggcatcc agtttttaat cttcagtggc
     9181 atgtgagatt ctccgaaatt aattaaagca atcacacaat tctctcggat accacctcgg
     9241 ttgaaactga caggtggttt gttacgcatg ctaatgcaaa ggagcctata tacctttggc
     9301 tcggctgctg taacagggaa tataaagggc agcataattt aggagtttag tgaacttgca
     9361 acatttacta ttttcccttc ttacgtaaat atttttcttt ttaattctaa atcaatcttt
     9421 ttcaattttt tgtttgtatt cttttcttgc ttaaatctat aactacaaaa aacacataca
     9481 g
//
