LOCUS       sequence-433204-        6147 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Three affibodies to HER2 linked in a chain for bacterial expression.
            SnoopTag and SpyTag allow isopeptide bond formation to their cognate
            Catcher.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6147)
  AUTHORS   Driscoll CL, Keeble AH, Howarth MR
  TITLE     SpyMask enables combinatorial assembly of bispecific binders.
  JOURNAL   Nat Commun. 2024 Mar 16;15(1):2403. doi: 10.1038/s41467-024-46599-9.
  PUBMED    38493197
REFERENCE   2  (bases 1 to 6147)
  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: "2024-03-16"; volume: "15(1):2403. doi"; pages: " 
            10.1038/s41467-024-46599-9"
COMMENT     Sequence Label: pET28a-SnoopTag-SpyTag-(AffiHER2)x3
FEATURES             Location/Qualifiers
     source          1..6147
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(857..886)
                     /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="ASMTGGQQMG"
     CDS             complement(911..949)
                     /codon_start=1
                     /product="peptide derived from the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyTag
                     /note="forms a covalent isopeptide bond with the SpyCatcher
                     protein (Zakeri et al., 2012)"
                     /translation="AHIVMVDAYKPTK"
     CDS             complement(980..1015)
                     /label=SnoopTag
     CDS             complement(1025..1042)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1052..1069)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1088..1110
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1125..1149
                     /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(1149..1168)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1150..1168)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1252..1271)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1477..1554
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1555..2637
                     /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(1574..1593)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3446..3637
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3631..3653)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3739..3878
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3794..3813
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3893..3910)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4064..4652)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4144..4163)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4763..4782)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4774..5589
                     /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             4774..5589
                     /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(4847..4866)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5681..6136)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5818..5839)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6030..6049
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcaa agcttttatt attctaatcc tttcggggcc tgcgcgtcgt
      181 tcagtttttt tgcttcggcc agcagattcg ctgactggga aggatcatca tacaaagagc
      241 ggataaaagc acgcttttgc tggttgttca ggtttggcag caatgcgatt tcccagtatg
      301 cgttccgcat ttctttattg aacttgttat ccacccccgg gtcgcggccc atctgctgac
      361 cgccagtcat actcgcccca ctaccttcac cgctgccccc cgagccctcc aggcctttcg
      421 gtgcctgggc gtcatttaat tttttagctt ccgctaacag gtttgcgctt tgactcggat
      481 catcatacag cgagcgaata aaggcgcgtt tctgttggtt attcaggttc ggcaacaggg
      541 cgatttccca gtaagcgttc cgcatttcct tgttaaattt attatctacg ccgggatcgc
      601 gtcccatctg ctggccaccc gtcatactag cgccactgcc ttcaccgcta ccgccggaac
      661 cctcgaggcc tttcggcgcc tgagcatcat ttagcttttt agcttctgct aaaaggttag
      721 cgctttggct tgggtcatcg tataacgacc ttatgaaagc ccttttctgt tgattgttta
      781 agttgggtaa aagagctatc tcccagtaag cgttcctcat ttctttgttg aatttgttgt
      841 ccacgcccgg atcccgaccc atttgctgtc caccagtcat gctagctccg gatcccgact
      901 caccactgcc cttcgtcggc ttgtaggcgt ccaccatcac gatgtgggcg ccgctgcctt
      961 cgccgctgcc gccgctgcct ttgttcactt taataaattc aatatcgccc agtttcccca
     1021 tatggctgcc gcgcggcacc aggccgctgc tgtgatgatg atgatgatgg ctgctgccca
     1081 tggtatatct ccttcttaaa gttaaacaaa attatttcta gaggggaatt gttatccgct
     1141 cacaattccc ctatagtgag tcgtattaat ttcgcgggat cgagatctcg atcctctacg
     1201 ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg
     1261 ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg
     1321 gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg
     1381 caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa
     1441 tgcaggagtc gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc
     1501 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa
     1561 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc
     1621 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg
     1681 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg
     1741 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg
     1801 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc
     1861 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg
     1921 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat
     1981 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc
     2041 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc
     2101 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat
     2161 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc
     2221 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg
     2281 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg
     2341 cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat
     2401 atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac
     2461 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca
     2521 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     2581 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     2641 caacgcaatt aatgtaagtt agctcactca ttaggcaccg ggatctcgac cgatgccctt
     2701 gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc
     2761 acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt
     2821 cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt
     2881 attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt
     2941 cggcgagaag caggccatta tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct
     3001 cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc
     3061 accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac
     3121 aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc
     3181 ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc
     3241 tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg
     3301 catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc
     3361 agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag
     3421 aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc
     3481 ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga
     3541 tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcagctg
     3601 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     3661 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     3721 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac
     3781 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccattg cggtgtgaaa
     3841 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca
     3901 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3961 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4021 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4081 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4141 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4201 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4261 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4321 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4381 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4441 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4501 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4561 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4621 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4681 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaac aataaaactg
     4741 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct
     4801 tgctctaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     4861 cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg
     4921 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     4981 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     5041 actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta
     5101 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     5161 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     5221 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     5281 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     5341 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     5401 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     5461 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     5521 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     5581 tttttctaag aattaattca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5641 aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt
     5701 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     5761 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     5821 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     5881 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     5941 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     6001 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     6061 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     6121 ccgctacagg gcgcgtccca ttcgcca
//
