LOCUS       sequence-449280-        5698 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial cell expression of SNAP-tag2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5698)
  AUTHORS   Kuhn S, Nasufovic V, Wilhelm J, Kompa J, de Lange EMF, Lin YH, 
            Egoldt C, Fischer J, Lennoi A, Tarnawski M, Reinstein J, Vlijm R, 
            Hiblot J, Johnsson K
  TITLE     SNAP-tag2 for faster and brighter protein labeling.
  JOURNAL   Nat Chem Biol. 2025 Jul 3. doi: 10.1038/s41589-025-01942-z.
  PUBMED    40610720
REFERENCE   2  (bases 1 to 5698)
  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: "Nat Chem 
            Biol. 2025 Jul 3. doi: 10.1038/s41589-025-01942-z."
COMMENT     Sequence Label: pET51b(+)_SNAP-tag2
FEATURES             Location/Qualifiers
     source          1..5698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1597..1788
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1782..1804)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1890..2029
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1945..1964
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2044..2061)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2215..2803)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2295..2314)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2884..2976
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2977..3045,3046..3837)
                     /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(3195..3214)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3952..4407)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4089..4110)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4301..4320
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4444..4491)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4487..4505
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4528..4557)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             complement(5110..5124)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             complement(5131..5154)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     RBS             5170..5192
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5207..5231
                     /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     complement(5231..5250)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5232..5250)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5338..5357)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5563..5640
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5641..5698,1..1025)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(5660..5679)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
       61 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
      121 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
      181 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
      241 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
      301 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
      361 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
      421 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
      481 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
      541 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
      601 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
      661 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
      721 ccgggctgcg cgttggtgcg gacatctcgg tagtgggata cgacgatacc gaagacagct
      781 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
      841 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
      901 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
      961 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     1021 agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg
     1081 atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactagc
     1141 atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag
     1201 cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg
     1261 acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg
     1321 aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg ctggctaccc
     1381 tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt gatttttctc
     1441 tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag taaccgggca
     1501 tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg tatcattacc
     1561 cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga aaaaaccgcc
     1621 cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa actcaacgag
     1681 ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc tgatgagctt
     1741 taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     1801 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     1861 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     1921 ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccatt
     1981 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc
     2041 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     2101 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     2161 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     2221 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     2281 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     2341 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     2401 gctttctcat agctcacgct gtaggtatct cagttcggtg tgggtcgttc gctccaagct
     2461 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     2521 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     2581 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     2641 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     2701 aaaaagagtt ggtagctctt ggtccggcaa acaaaccacc gctggtagcg gtggtttttt
     2761 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     2821 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgac
     2881 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     2941 gataaatgct tcaatcatga ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     3001 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     3061 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     3121 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     3181 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     3241 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     3301 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     3361 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     3421 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     3481 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     3541 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     3601 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     3661 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     3721 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     3781 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     3841 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     3901 aggatctagg tgaagatcct ttttgataat ctcatgccat cgtgccgcca gaaattgtaa
     3961 acgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     4021 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     4081 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     4141 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     4201 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     4261 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     4321 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     4381 cgcttaatgc gccgctacag ggcgcgtccc attcgccaat ccggatatag ttcctccttt
     4441 cagcaaaaaa cccctcaaga cccgtttaga ggccccaagg ggttatgcta gttattgctc
     4501 agcggtggca gcagcctagg ttaattagtg gtggtgatgg tgatgatggt ggtgatgagc
     4561 ggaaattgag gagaagcccg gagcgcccag gcctggttta cccagacggt gaccttcgtg
     4621 cgccagcagc cattctttca cgtacagagg gccttcgtag ccacctacgt cgccgtcgcc
     4681 ctgaaccaca cggtggcacg ggatcagaat tggcaccgga ttaccacgca gagcggtttt
     4741 caccgccgca gttgccgctg ggttgccagc cagcgccgcc agctgggaat aggagatcac
     4801 ctcgccgaat ttcactactt tcagcagttt ccacagtacc tgacgggtaa aggattcctg
     4861 ctgaaatacc gggtgatgca gggccggtac agggaactct tcaatcgcct ctggctggtg
     4921 aaaatatgcg ttcagccatg cggtagcctg catcagcggc tccgggcccg gaggaccctg
     4981 ctcgccctga ccattgccca cgaaatagat gcggtgcagg ccctgctcgc aaccgctcag
     5041 cagcagttta cccagcgggc tgtcataggt agtgcgtttc atttcgcagt ctttatccat
     5101 atgcggtacc ttgtcgtcgt catctgcacc cttttcgaac tgcgggtggc tccagcttgc
     5161 catggtatat ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg
     5221 ctcacaattc ccctatagtg agtcgtatta atttcgcggg atcgagatcg atctcgatcc
     5281 tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct
     5341 atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt
     5401 gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct
     5461 tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct
     5521 tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc
     5581 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
     5641 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccg
//
