LOCUS       sequence-340789-        6330 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses spNW15 in Bl21.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6330)
  AUTHORS   Zhang S, Ren Q, Novick SJ, Strutzenberg TS, Griffin PR, Bao H
  TITLE     One-step construction of circularized nanodiscs using 
            SpyCatcher-SpyTag.
  JOURNAL   Nat Commun. 2021 Sep 14;12(1):5451. doi: 10.1038/s41467-021-25737-7.
  PUBMED    34521837
REFERENCE   2  (bases 1 to 6330)
  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 
            Commun."; date: "2021-09-14"; volume: "12(1):5451. doi"; pages: " 
            10.1038/s41467-021-25737-7"
COMMENT     Sequence Label: spNW15
FEATURES             Location/Qualifiers
     source          1..6330
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     961..980
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1000..1077)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1283..1302
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1386..1405
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1386..1404
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1405..1429
                     /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             1444..1466
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1485..1502
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1512..1529
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1533..1565
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             1584..1922
                     /codon_start=1
                     /product="improved SpyCatcher variant that shows rapid 
                     reaction with SpyTag002 (Keeble et al., 2017)"
                     /label=SpyCatcher002
                     /translation="VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATME
                     LRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG
                     EATKGDAHT"
     CDS             2520..2561
                     /codon_start=1
                     /product="improved SpyTag variant that shows rapid reaction
                     with SpyCatcher002 (Keeble et al., 2017)"
                     /label=SpyTag002
                     /translation="VPTIVMVDAYKRYK"
     CDS             2580..2597
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2650..2668)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2664..2711
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2748..3203
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2835..2854)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3045..3066
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3295..4110)
                     /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             complement(3295..4110)
                     /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     4018..4037
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4102..4121
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4232..4820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4721..4740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4974..4991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5006..5145
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5071..5090)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5231..5253
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5247..5438)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6247..6330,1..999))
                     /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"
ORIGIN
        1 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
       61 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
      121 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
      181 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
      241 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
      301 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
      361 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
      421 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
      481 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
      541 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
      601 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
      661 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
      721 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
      781 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
      841 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
      901 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
      961 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     1021 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     1081 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     1141 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     1201 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     1261 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     1321 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     1381 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
     1441 aattttgttt aactttaaga aggagatata ccatgggcag cagccatcat catcatcatc
     1501 acagcagcgg cctggtgccg cgcggcagcc atatggctag catgactggt ggacagcaaa
     1561 tgggtcgcgg atccggcgcc atggtaacca ccttatcagg tttatcaggt gagcaaggtc
     1621 cgtccggtga tatgacaact gaagaagata gtgctaccca tattaaattc tcaaaacgtg
     1681 atgaggacgg ccgtgagtta gctggtgcaa ctatggagtt gcgtgattca tctggtaaaa
     1741 ctattagtac atggatttca gatggacatg tgaaggattt ctacctgtat ccaggaaaat
     1801 atacatttgt cgaaaccgca gcaccagacg gttatgaggt agcaactgct attaccttta
     1861 cagttaatga gcaaggtcag gttactgtaa atggcgaagc aactaaaggt gacgctcata
     1921 ctagtacctt ctccaaactg cgcgaacagc tgggtccggt tacccaggaa ttctgggaca
     1981 acctggaaaa ggagacggaa ggcctgcgtc aagaaatgag caaagatctg gaagaagtta
     2041 aagcgaaagt ccagccgtac ctggatgact ttcagaaaaa atggcaagaa gaaatggagt
     2101 tataccgcca gaaagttgaa ccgctgcgtg cagaactgca ggaaggcgct cgtcagaaac
     2161 tgcacgagtt acaagaaaaa ctgagtccgc tgggagagga aatgcgcgac cgtgcccgtg
     2221 cacatgttga tgcactgcgt acgcatctgg caccgtattc cgacgaactg cgtcaacgcc
     2281 tggccgcccg cctggaagcg ttaaaggaaa atggcggtgc ccgtctggcc gaatatcacg
     2341 cgaaagccac ggaacatctg agcacgctgt ctgaaaaagc caagcctgcc ctggaagatc
     2401 tgcgccaggg gctgctgccg gttctggaaa gctttaaagt cagtttcctg tccgcgctgg
     2461 aagaatatac caaaaaactg aatacccagc tgccgggtac cggtgcagct gccctggaag
     2521 tgcctactat cgtgatggtg gacgcctaca agcgttacaa gtaagcggcc gcactcgagc
     2581 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     2641 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     2701 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     2761 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2821 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2881 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2941 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     3001 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3061 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     3121 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     3181 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     3241 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     3301 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     3361 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     3421 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     3481 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     3541 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     3601 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     3661 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     3721 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     3781 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     3841 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     3901 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     3961 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     4021 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     4081 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     4141 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     4381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     4561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4981 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     5041 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     5101 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     5161 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     5221 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     5281 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     5341 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     5401 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     5461 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     5521 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     5581 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     5641 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     5701 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     5761 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     5821 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     5881 aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg
     5941 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     6001 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     6061 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     6121 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     6181 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     6241 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     6301 ggccaacgcg cggggagagg cggtttgcgt
//
