LOCUS       sequence-226326-        2672 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of proteins with C-terminal Spot-Tag in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2672)
  TITLE     ChromoTek Spot-tag plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 2672)
  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            "Unpublished"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: pSpot2
FEATURES             Location/Qualifiers
     source          1..2672
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             141..176
                     /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(229..247)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     primer_bind     complement(311..328)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      481..567
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      659..686
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        705..796
                     /gene="bla"
                     /label=AmpR promoter
     CDS             797..1612
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             797..1612
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(870..889)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1796..2384
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2285..2304
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg gatccgaatt cgagctccgt cgacaagctt
      121 gcggccgcac tcgagctagc ccggatcgcg tgcgcgcagt ctctcactgg agcagctaag
      181 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
      241 aactagcata accccttggg gcaaacggtc tccagcttgg ctgttttggc ggatgagaga
      301 agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt
      361 tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac
      421 gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat
      481 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
      541 gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa
      601 cggcccggag ggtggcgggc aggacgcccg ccataaactg ccaggcatca aattaagcag
      661 aaggccatcc tgacggatgg cctttttgcg tttctacaaa ctcttttgtt tatttttcta
      721 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
      781 ttgaaaaagg aagagtatga gccatattca acgggaaacg tcttgctcta ggccgcgatt
      841 aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca
      901 atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa
      961 acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct
     1021 gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg
     1081 gttactcacc actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga
     1141 ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc
     1201 tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg
     1261 aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt
     1321 tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac
     1381 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat
     1441 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg
     1501 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa
     1561 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aataagcatt
     1621 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     1681 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     1741 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     1801 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     1861 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     1921 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     1981 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     2041 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     2101 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     2161 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     2221 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2281 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2341 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2401 cctttttacg gttcctggcc ttttgctggc cttttgctca catgcgcaac gcaattaatg
     2461 taagttagcg cgaattgatc tggtttgaca gcttatcatc gactgcacgg tgcaccaatg
     2521 cttctggcgt caggcagcca tcggaagctg tggtatggct gtgcaggtcg taaatcactg
     2581 cataattcgt gtcgctcaag gcgcactccc gttctggata atgttttttg cgccgacatc
     2641 ataacggttc tggcaaatat tctgaaatga gc
//
