LOCUS       sequence-421901-        4172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of VHH with C-terminal his and Flag tags.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4172)
  AUTHORS   Eyssen LE, Ramadurai S, Abdelkarim S, Buckle I, Cornish K, Lin H, 
            Jones AK, Stephens GJ, Owens RJ
  TITLE     From Llama to Nanobody: A Streamlined Workflow for the Generation of
            Functionalised VHHs.
  JOURNAL   Bio Protoc. 2024 Mar 20;14(6):e4962. doi: 10.21769/BioProtoc.4962. 
            eCollection 2024 Mar 20.
  PUBMED    38841291
REFERENCE   2  (bases 1 to 4172)
  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: "Bio 
            Protoc."; date: "2024-03-20"; volume: "14(6):e4962. doi"; pages: " 
            10.21769/BioProtoc.4962. eCollection 2024 Mar 20"
COMMENT     Sequence Label: pOPINVHH_his_flag
FEATURES             Location/Qualifiers
     source          1..4172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..125
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(126..194,195..986)
                     /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     complement(344..363)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1157..1745
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1646..1665
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1910..1931
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1946..1951,1952..1969,1970..1976)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1984..2000
                     /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(2109..2126)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2109..2125)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2118..2140)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2280..2297
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2298..2321
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2344..2373
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     rep_origin      complement(3738..4166)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3848..3869)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4060..4079
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
       61 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
      121 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
      181 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
      241 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
      301 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
      361 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
      421 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
      481 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
      541 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
      601 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
      661 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
      721 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
      781 tgcgctcggc gcttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
      841 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
      901 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
      961 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     1021 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     1081 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1141 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1201 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1261 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     1321 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1381 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1441 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1501 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     1561 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1621 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     1681 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     1741 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     1801 acatgcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     1861 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     1921 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     1981 gaattgtgag cggataacaa tttgaattca aggagacagt cataatgaaa tacctattgc
     2041 ctacggcggc cgctggattg ttattactcg cggcccagcc ggccatggat gaccatgatt
     2101 aaggattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     2161 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     2221 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcca tcctcaggcc
     2281 accatcacca tcaccatgac tacaaagacg atgacgacaa gtaataacca ccatcatggc
     2341 gcagaacaaa aactcatctc agaagaggat ctgtcttagg ccgaaactgt tgaaagttgt
     2401 ttagcaaaac ctcatacaga aaattcattt actaacgtct ggaaagacga caaaacttta
     2461 gatcgttacg ctaactatga gggctgtctg tggaatgcta caggcgttgt ggtttgtact
     2521 ggtgacgaaa ctcagtgtta cggtacatgg gttcctattg ggcttgctat ccctgaaaat
     2581 gagggtggtg gctctgaggg tggcggttct gagggtggcg gttctgaggg tggcggtact
     2641 aaacctcctg agtacggtga tacacctatt ccgggctata cttatatcaa ccctctcgac
     2701 ggcacttatc cgcctggtac tgagcaaaac cccgctaatc ctaatccttc tcttgaggag
     2761 tctcagcctc ttaatacttt catgtttcag aataataggt tccgaaatag gcagggtgca
     2821 ttaactgttt atacgggcac tgttactcaa ggcactgacc ccgttaaaac ttattaccag
     2881 tacactcctg tatcatcaaa agccatgtat gacgcttact ggaacggtaa attcagagac
     2941 tgcgctttcc attctggctt taatgaggat ccattcgttt gtgaatatca aggccaatcg
     3001 tctgacctgc ctcaacctcc tgtcaatgct ggcggcggct ctggtggtgg ttctggtggc
     3061 ggctctgagg gtggcggctc tgagggtggc ggttctgagg gtggcggctc tgagggtggc
     3121 ggttccggtg gcggctccgg ttccggtgat tttgattatg aaaaaatggc aaacgctaat
     3181 aagggggcta tgaccgaaaa tgccgatgaa aacgcgctac agtctgacgc taaaggcaaa
     3241 cttgattctg tcgctactga ttacggtgct gctatcgatg gtttcattgg tgacgtttcc
     3301 ggccttgcta atggtaatgg tgctactggt gattttgctg gctctaattc ccaaatggct
     3361 caagtcggtg acggtgataa ttcaccttta atgaataatt tccgtcaata tttaccttct
     3421 ttgcctcagt cggttgaatg tcgcccttat gtctttggcg ctggtaaacc atatgaattt
     3481 tctattgatt gtgacaaaat aaacttattc cgtggtgtct ttgcgtttct tttatatgtt
     3541 gccaccttta tgtatgtatt ttcgacgttt gctaacatac tgcgtaataa ggagtcttaa
     3601 gctagctaac agtcctatga atcaactact tagatggtat tagtgacctg taacagagca
     3661 ttagcgcaag gtgatttttg tcttcttgcg ctaatttttt gtcatcaaac ctgtcgcact
     3721 ccttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     3781 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     3841 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     3901 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     3961 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     4021 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     4081 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     4141 gcttaatgcg ccgctacagg gcgcgtcagg tg
//
