LOCUS       sequence-220575-        2673 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 2673)
  TITLE     ChromoTek Spot-tag plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 2673)
  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            "Unpublished"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..2673
                     /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        706..797
                     /gene="bla"
                     /label=AmpR promoter
     CDS             798..1613
                     /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             798..1613
                     /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(871..890)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1797..2385
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2286..2305
                     /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 ctctttttgt ttatttttct
      721 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
      781 attgaaaaag gaagagtatg agccatattc aacgggaaac gtcttgctct aggccgcgat
      841 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
      901 aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga
      961 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
     1021 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
     1081 ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa gaatatcctg
     1141 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
     1201 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac
     1261 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
     1321 ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca
     1381 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
     1441 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
     1501 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
     1561 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taataagcat
     1621 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     1681 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     1741 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     1801 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     1861 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     1921 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     1981 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     2041 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     2101 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     2161 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     2221 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     2281 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2341 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2401 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgcgcaa cgcaattaat
     2461 gtaagttagc gcgaattgat ctggtttgac agcttatcat cgactgcacg gtgcaccaat
     2521 gcttctggcg tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact
     2581 gcataattcg tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat
     2641 cataacggtt ctggcaaata ttctgaaatg agc
//
