LOCUS       sequence-390537-        4539 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid for anti-GFP nanobody with an 
            N-terminal His-tag and biotin acceptor peptide (Avi).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4539)
  AUTHORS   Stevens TA, Tomaleri GP, Hazu M, Wei S, Nguyen VN, DeKalb C, 
            Voorhees RM, Pleiner T
  TITLE     A nanobody-based strategy for rapid and scalable purification of 
            human protein complexes.
  JOURNAL   Nat Protoc. 2024 Jan;19(1):127-158. doi: 10.1038/s41596-023-00904-w.
            Epub 2023 Nov 16.
  PUBMED    37974029
REFERENCE   2  (bases 1 to 4539)
  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"; doi: 
            "10.1038/s41596-023-00904-w"; journalName: "Nat Protoc"; date: 
            "2024-01"; volume: "19"; issue: "1"; pages: "127-158"
COMMENT     Sequence Label: pTS117 His14-Avi-45xGS-anti GFP nanobody
FEATURES             Location/Qualifiers
     source          1..4539
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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)."
     protein_bind    62..78
                     /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)."
     RBS             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             205..249
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             391..732
                     /codon_start=1
                     /product="GFP-binding fragment of a single-chain camelid 
                     antibody (Rothbauer et al., 2008)"
                     /label=GFP nanobody
                     /translation="VQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKERE
                     WVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYW
                     GQGTQVTVSS"
     CDS             744..761
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      784..878
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(926..945)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             937..1752
                     /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             937..1752
                     /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(1010..1029)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1971..2057
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2150..3232)
                     /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     3194..3213
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3233..3310)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(3394..3416)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3502..3642
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3557..3576
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3657..3674)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3828..4416)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3908..3927)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4507..4525
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aaccatgagc
      121 aagcatcacc atcattcagg ccatcaccat accggacacc accatcattc aggcagtcat
      181 caccataccg gcggatcatc cggaggtctg aatgatatct ttgaagcaca aaagattgaa
      241 tggcatgaag gctctggatc tgcaactggt tcaggaggca gtgggggttc cggtacaccg
      301 ggcagttcta cgagtggcac ctctgcagga tccggttctg gtggtagcgg tggttcagga
      361 ggcagtgggg gttccactag taccgctcag gtgcagctag ttgaaagcgg cggtgcgctg
      421 gttcagccgg gtggtagcct gcgtctgagc tgtgcggcga gcggctttcc ggtgaaccgt
      481 tatagcatgc gctggtatcg tcaggcaccg ggcaaagaac gtgaatgggt ggcaggcatg
      541 agcagcgcgg gtgatcgtag cagctatgaa gatagcgtga aaggccgttt taccattagc
      601 cgtgatgatg cgcgtaacac cgtgtatctg cagatgaaca gcctgaaacc ggaagatacc
      661 gcggtgtatt attgcaacgt gaacgtgggc tttgaatatt ggggccaggg cacacaggtt
      721 acagttagta gctgacggcc gcacatcacc atcaccatca ctaagcttaa ttagctgagc
      781 ttggactcct gttgatagat ccagtaatga cctcagaact ccatctggat ttgttcagaa
      841 cgctcggttg ccgccgggcg ttttttattg gtgagaatcc aagctagcca tgaaaataaa
      901 ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg
      961 tcttgctcta ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg
     1021 gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat
     1081 gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag
     1141 atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc
     1201 cgtactcctg atgatgcttg gttactcacg actgcgatcc ccggcaaaac agcattccag
     1261 gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg
     1321 cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt
     1381 ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac
     1441 gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcacaaact tttgccattc
     1501 tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag
     1561 gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat
     1621 cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt
     1681 caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat
     1741 gagtttttct aagaattaat tcatgggcaa atattatacg caaggcgaca aggtgctgat
     1801 gccgctggcg attcaggttc atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct
     1861 taatgaatta caacagtact gcgatgagtg gcagggcggg gcgtaatttt tttaaggcag
     1921 ttattggtgc ccttaaacgc ctggggtaat gactctctag cttgaggcat caaataaaac
     1981 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     2041 tcctgagtag gacaaatccg ccctctagat tacgtgcagt cgatgataag ctgtcaaaca
     2101 tgagaattgt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     2161 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     2221 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     2281 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     2341 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     2401 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     2461 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     2521 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     2581 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     2641 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     2701 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     2761 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     2821 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     2881 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     2941 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     3001 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     3061 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     3121 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     3181 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     3241 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcaccatt
     3301 cgatggtgtc ggaatttcgg gcagcgttgg gtcctggcca cgggtgcgca tgatctagag
     3361 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     3421 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     3481 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     3541 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
     3601 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg
     3661 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     3721 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     3781 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3841 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3901 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3961 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4021 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4081 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4141 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4201 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4261 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4321 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4381 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4441 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gacattaacc
     4501 tataaaaata ggcgtatcac gaggcccttt cgtcttcac
//
