LOCUS       sequence-421924-        4842 bp    DNA     circular SYN 14-APR-2025
DEFINITION  For expression of VHH with C-terminal snap protein and his tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4842)
  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 4842)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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_snap_his
FEATURES             Location/Qualifiers
     source          1..4842
                     /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)."
     sig_peptide     2025..2090
                     /codon_start=1
                     /gene="pelB (fragment)"
                     /product="leader peptide for secretion"
                     /label=pelB signal sequence
                     /translation="MKYLLPTAAAGLLLLAAQPAMA"
     protein_bind    2130..2151
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2166..2171,2172..2189,2190..2196)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2204..2220
                     /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     2228..2244
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2228..2244
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2240..2413
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
     primer_bind     complement(2260..2277)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2260..2276)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2269..2291)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2974..2991
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3014..3043
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     rep_origin      complement(4408..4836)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4518..4539)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4730..4749
                     /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 ggccatggcg tggacccgac
     2101 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     2161 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     2221 tttcacacag gaaacagcta tgaccatgat tacggattca ctggccgtcg ttttacaacg
     2281 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     2341 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     2401 cctgaatggc gaatggcgcc atcctcaggc gacaaggact gcgaaatgaa gcgtactact
     2461 ctggatagcc cgcttggtaa gttggagctg tccggctgcg agcaagggct tcatgagatc
     2521 aaattgttgg gtaagggtac aagtgctgcg gacgctgtag aagtccctgc acccgccgct
     2581 gtgcttggag gtcccgaacc tttaatgcag gcgaccgcct ggctgaacgc ctactttcat
     2641 cagccggagg ctatcgaaga atttcccgtc ccggctctgc accaccctgt gtttcaacaa
     2701 gaaagtttta ctcgtcaggt cctgtggaaa ctgcttaagg tagttaaatt tggagaggta
     2761 atctcgtatc aacagttagc ggccttagct ggcaacccgg cagccacagc agctgtaaaa
     2821 acggcgctgt ctggaaaccc tgtgccaatt cttattcctt gtcatcgtgt tgtatcaagt
     2881 tcaggtgctg tgggcggcta cgaaggagga ttagccgtaa aggaatggtt attggcgcac
     2941 gaggggcacc gtcttggaaa gccgggtttg ggtcaccatc accatcacca ttaataacca
     3001 ccatcatggc gcagaacaaa aactcatctc agaagaggat ctgtcttagg ccgaaactgt
     3061 tgaaagttgt ttagcaaaac ctcatacaga aaattcattt actaacgtct ggaaagacga
     3121 caaaacttta gatcgttacg ctaactatga gggctgtctg tggaatgcta caggcgttgt
     3181 ggtttgtact ggtgacgaaa ctcagtgtta cggtacatgg gttcctattg ggcttgctat
     3241 ccctgaaaat gagggtggtg gctctgaggg tggcggttct gagggtggcg gttctgaggg
     3301 tggcggtact aaacctcctg agtacggtga tacacctatt ccgggctata cttatatcaa
     3361 ccctctcgac ggcacttatc cgcctggtac tgagcaaaac cccgctaatc ctaatccttc
     3421 tcttgaggag tctcagcctc ttaatacttt catgtttcag aataataggt tccgaaatag
     3481 gcagggtgca ttaactgttt atacgggcac tgttactcaa ggcactgacc ccgttaaaac
     3541 ttattaccag tacactcctg tatcatcaaa agccatgtat gacgcttact ggaacggtaa
     3601 attcagagac tgcgctttcc attctggctt taatgaggat ccattcgttt gtgaatatca
     3661 aggccaatcg tctgacctgc ctcaacctcc tgtcaatgct ggcggcggct ctggtggtgg
     3721 ttctggtggc ggctctgagg gtggcggctc tgagggtggc ggttctgagg gtggcggctc
     3781 tgagggtggc ggttccggtg gcggctccgg ttccggtgat tttgattatg aaaaaatggc
     3841 aaacgctaat aagggggcta tgaccgaaaa tgccgatgaa aacgcgctac agtctgacgc
     3901 taaaggcaaa cttgattctg tcgctactga ttacggtgct gctatcgatg gtttcattgg
     3961 tgacgtttcc ggccttgcta atggtaatgg tgctactggt gattttgctg gctctaattc
     4021 ccaaatggct caagtcggtg acggtgataa ttcaccttta atgaataatt tccgtcaata
     4081 tttaccttct ttgcctcagt cggttgaatg tcgcccttat gtctttggcg ctggtaaacc
     4141 atatgaattt tctattgatt gtgacaaaat aaacttattc cgtggtgtct ttgcgtttct
     4201 tttatatgtt gccaccttta tgtatgtatt ttcgacgttt gctaacatac tgcgtaataa
     4261 ggagtcttaa gctagctaac agtcctatga atcaactact tagatggtat tagtgacctg
     4321 taacagagca ttagcgcaag gtgatttttg tcttcttgcg ctaatttttt gtcatcaaac
     4381 ctgtcgcact ccttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     4441 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     4501 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     4561 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     4621 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     4681 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     4741 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     4801 cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tg
//
