LOCUS       sequence-404736-        6213 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of SARS-CoV-2 Receptor Binding Domain with 
            peptide tags for covalent bond formation .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6213)
  AUTHORS   Keeble AH, Wood DP, Howarth M
  TITLE     Design and Evolution of Enhanced Peptide-Peptide Ligation for 
            Modular Transglutaminase Assembly.
  JOURNAL   Bioconjug Chem. 2023 Jun 8. doi: 10.1021/acs.bioconjchem.3c00122.
  PUBMED    37289810
REFERENCE   2  (bases 1 to 6213)
  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: "Bioconjug 
            Chem. 2023 Jun 8. doi: 10.1021/acs.bioconjchem.3c00122."
COMMENT     Sequence Label: pcDNA3.1 RBD-DogTag2-SpyTag
FEATURES             Location/Qualifiers
     source          1..6213
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    203..324
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(240..259)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     294..313
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(373..389)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(373..389)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(386..408)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    397..413
                     /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)."
     promoter        complement(join(421..427,428..445,446..451))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    466..487
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(604..621)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(775..1363)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(855..874)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1534..2325,2326..2394))
                     /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     2157..2176
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2395..2499)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2574..2593)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2765..3144
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3145..3348
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3299..3319
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3393..3412
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3393..3411
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(3865..3882)
                     /codon_start=1
                     /product="tetracysteine peptide that binds biarsenical 
                     labeling reagents"
                     /label=tetracysteine tag
                     /translation="CCPGCC"
     CDS             4158..4226
                     /codon_start=1
                     /product="peptide derived from the RrgA adhesin of 
                     Streptococcus pneumoniae"
                     /label=DogTag
                     /note="forms a covalent bond with the DogCatcher protein 
                     when inserted into an intra-domain protein loop (Keeble et 
                     al., 2022)"
                     /translation="DIPATYEFTDGKHYITNEPLPPK"
     CDS             4245..4283
                     /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"
     primer_bind     complement(4355..4372)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4361..4585
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4631..5059
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4718..4737)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4928..4949
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5068..5088)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5233..5367
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5294..5313
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             join(5448..6213,1..29)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5502..5521)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6112..6131
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
       61 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
      121 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
      181 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
      241 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
      301 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
      361 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
      421 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
      481 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
      541 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
      601 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
      661 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
      721 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
      781 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
      841 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
      901 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
      961 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     1021 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     1081 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     1141 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     1201 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     1261 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     1321 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     1381 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     1441 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     1501 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     1561 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     1621 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     1681 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     1741 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     1801 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     1861 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     1921 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     1981 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     2041 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     2101 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     2161 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     2221 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     2281 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     2341 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     2401 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     2461 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     2521 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
     2581 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
     2641 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
     2701 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
     2761 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
     2821 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     2881 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     2941 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     3001 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     3061 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     3121 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
     3181 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     3241 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     3301 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
     3361 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
     3421 gctggctagc gtttaaacgg gccctctaga ctcgagcggc cgccaccatg aacacccaga
     3481 tcctggtgtt cgccctgatc gccatcattc ccaccaacgc cgacaagatc ggatctggat
     3541 ccaacatcac caatctgtgc cccttcggcg aggtgttcaa tgccacaaga tttgccagcg
     3601 tgtacgcctg gaaccggaag agaatcagca actgcgtggc cgactacagc gtgctgtaca
     3661 atagcgccag cttcagcacc ttcaagtgct acggcgtgtc ccctaccaag ctgaacgacc
     3721 tgtgcttcac caatgtgtac gccgacagct tcgtgatcag aggcgacgaa gttcggcaga
     3781 tcgctcctgg acagacaggc aagatcgccg attacaacta caagctgccc gacgacttca
     3841 ccggctgcgt gatcgcctgg aatagcaaca acctggacag caaagtcggc ggcaactaca
     3901 actacctgta ccggctgttc cggaagtcca acctgaagcc tttcgagcgg gacatcagca
     3961 ccgagatcta tcaggccggc agcacccctt gtaatggcgt ggaaggcttc aactgctact
     4021 tcccactgca gtcctacggc ttccagccta caaatggcgt cggctaccag ccttacagag
     4081 tggtggtgct gtccttcgaa ctgctgcatg cccctgctac cgtgtgcggc cctaaaaaaa
     4141 ccggaggagg tggctctgat attccggcta catacgaatt caccgatggt aaacattata
     4201 tcaccaatga accgctgccg ccgaaaggta gcggcggatc tggagcccac atcgtgatgg
     4261 ttgatgccta caagcccacc aaatagtgaa ttccaccaca ctggactagt ggatccgagc
     4321 tcggtaccaa gcttaagttt aaaccgctga tcagcctcga ctgtgccttc tagttgccag
     4381 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     4441 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     4501 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     4561 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     4621 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4681 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     4741 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4801 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     4861 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     4921 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     4981 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     5041 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     5101 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     5161 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     5221 cagcaaccat agtcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     5281 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     5341 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     5401 tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     5461 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     5521 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     5581 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     5641 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     5701 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     5761 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     5821 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     5881 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     5941 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     6001 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     6061 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     6121 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     6181 gtatcgccgc tcccgattcg cagcgcatcg cct
//
