LOCUS       sequence-473212-        5654 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express SpyCatcher.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5654)
  AUTHORS   Gutu N, Peng Y, Renn D, Rueping M
  TITLE     Nanobodies for NETosis Tracking and Visualization.
  JOURNAL   Curr Protoc. 2025 Oct;5(10):e70213. doi: 10.1002/cpz1.70213.
  PUBMED    41090551
REFERENCE   2  (bases 1 to 5654)
  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: "Curr 
            Protoc."; date: "2025-10"; volume: "5(10):e70213. doi"; pages: " 
            10.1002/cpz1.70213"
COMMENT     Sequence Label: pET29b_His-TEV-SpyCatcher003-C
FEATURES             Location/Qualifiers
     source          1..5654
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             7..24
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(77..95)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      91..138
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      175..630
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(262..281)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     472..493
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(722..1537)
                     /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(722..1537)
                     /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     1445..1464
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1529..1548
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1659..2247
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2148..2167
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2401..2418
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2433..2572
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2498..2517)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2658..2680
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2674..2865)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3674..4756)
                     /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     4718..4737
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4757..4834)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5040..5059
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5147..5166
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5147..5165
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5166..5190
                     /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             5205..5227
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5247..5264
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5286..5306
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
ORIGIN
        1 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
       61 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
      121 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
      181 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
      241 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
      301 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
      361 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
      421 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
      481 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
      541 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
      601 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
      661 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
      721 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
      781 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
      841 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
      901 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
      961 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     1021 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     1081 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     1141 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     1201 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     1261 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     1321 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     1381 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     1441 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     1501 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     1561 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     1621 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     1681 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     1741 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     1801 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     1861 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     1921 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     1981 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     2041 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2101 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2161 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     2221 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2281 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     2341 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     2401 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     2461 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     2521 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     2581 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     2641 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     2701 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     2761 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     2821 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     2881 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     2941 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     3001 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     3061 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     3121 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     3181 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     3241 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     3301 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     3361 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     3421 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     3481 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     3541 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     3601 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     3661 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3721 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     3781 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     3841 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     3901 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg
     3961 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     4021 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     4081 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     4141 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     4201 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     4261 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     4321 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     4381 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     4441 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     4501 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     4561 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     4621 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     4681 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     4741 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     4801 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     4861 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     4921 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     4981 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     5041 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     5101 atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg cgaaattaat acgactcact
     5161 ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag
     5221 aaggagatat acatatgtct tattaccatc atcaccacca ccacgattac gacataccga
     5281 cgactgagaa cttgtacttc caaggggcta tggtgactac actgtccggc ctgtcaggtg
     5341 aacaaggccc aagtggggac atgactaccg aagaggactc ggcaacgcat ataaagttct
     5401 caaagcgcga cgaagatggt cgtgaacttg cgggtgctac catggaattg agagactgtt
     5461 cagggaagac aatatccaca tggataagtg acggtcatgt aaaggacttc tatttatacc
     5521 caggtaagta cacatttgtc gagactgcag cacctgacgg ctatgaggtt gctactccta
     5581 tcgagttcac ggtgaatgaa gacggtcagg tgacggtaga cggcgaggcg acagaaggcg
     5641 acgcacacac ttag
//
