LOCUS       sequence-433378-        5646 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses nanoHER2-SpyTag003 in bacterial cells. nanoHER2 is an 
            anti-HER2 nanobody..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5646)
  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 5646)
  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-nanoHER2-SpyTag003
FEATURES             Location/Qualifiers
     source          1..5646
                     /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(143..160)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(170..217)
                     /codon_start=1
                     /product="improved SpyTag variant that reacts with 
                     SpyCatcher003 at a rate approaching the diffusion limit 
                     (Keeble et al., 2019)"
                     /label=SpyTag003
                     /translation="RGVPHIVMVDAYKRYK"
     RBS             587..609
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    624..648
                     /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(648..667)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(649..667)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(751..770)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        976..1053
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1054..2136
                     /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(1073..1092)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2945..3136
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3130..3152)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3238..3377
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3293..3312
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3392..3409)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3563..4151)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3643..3662)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4262..4281)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4273..5088
                     /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             4273..5088
                     /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(4346..4365)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5180..5635)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5317..5338)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5529..5548
                     /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 cggatcttat taatggtgat ggtgatggtg acctgaaccc ttgtaacgct
      181 tgtaggcgtc caccatcacg atatgaggca cgccacgaga gctaaccgtc acttgggtac
      241 cctggcccca ataattgttg tcaatgaatt gcggtttcac ccagcgggca ttgcagtaat
      301 agaccgcggt atcttccggt ttcagactgt tcatctgcag gtacaccgta ttttttgcgt
      361 tgtcgcgcga gatggtaaaa cggcctttga ctgaatcagc ataattcgtg gtaccgccgg
      421 agttaatact tgcaaccatt tcacgttgtt tacccgggct ctgacggtac caacccagtg
      481 cataacgcat aaacgtgatg cccgaggtag cacaggacag acgcagtgag ccgcccgctt
      541 gaaccagacc accgcccgat tccaccagtt gaacttccat ggtatatctc cttcttaaag
      601 ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc tatagtgagt
      661 cgtattaatt tcgcgggatc gagatctcga tcctctacgc cggacgcatc gtggccggca
      721 tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag
      781 atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc
      841 ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg
      901 tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag
      961 agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag
     1021 cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc
     1081 gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac
     1141 gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc
     1201 aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc
     1261 agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa
     1321 ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg
     1381 gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat
     1441 gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat
     1501 gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg
     1561 ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta
     1621 agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa
     1681 attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc
     1741 atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg
     1801 gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg
     1861 gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc
     1921 aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag
     1981 ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc
     2041 ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     2101 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta
     2161 gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca acccagtcag
     2221 ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat
     2281 catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg aggaccgctt
     2341 tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct tgcacgccct
     2401 cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat
     2461 cgccggcatg gcggccccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct
     2521 aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg
     2581 aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt
     2641 ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat
     2701 ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg
     2761 ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt
     2821 accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat
     2881 cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc
     2941 atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac
     3001 attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga
     3061 atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga
     3121 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     3181 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc
     3241 agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca
     3301 gagcagattg tactgagagt gcaccattgc ggtgtgaaat accgcacaga tgcgtaagga
     3361 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     3421 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     3481 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3541 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3601 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3661 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3721 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3781 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     3841 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     3901 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     3961 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     4021 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4081 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4141 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4201 actcacgtta agggattttg gtcatgaaca ataaaactgt ctgcttacat aaacagtaat
     4261 acaaggggtg ttatgagcca tattcaacgg gaaacgtctt gctctaggcc gcgattaaat
     4321 tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca
     4381 ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat
     4441 ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg
     4501 gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta
     4561 ctcaccactg cgatccccgg gaaaacagca ttccaggtat tagaagaata tcctgattca
     4621 ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt
     4681 tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg
     4741 aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa
     4801 caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat
     4861 ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat
     4921 gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc
     4981 ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct
     5041 gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaaga attaattcat
     5101 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     5161 tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     5221 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     5281 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     5341 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     5401 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     5461 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     5521 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     5581 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
     5641 tcgcca
//
