LOCUS       sequence-298984-        6460 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NB-alpha-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6460)
  AUTHORS   Iwaki T, Hara K, Umemura K
  TITLE     Nanobody production can be simplified by direct secretion from 
            Escherichia coli.
  JOURNAL   Protein Expr Purif. 2020 Feb 13;170:105607. doi: 
            10.1016/j.pep.2020.105607.
  PUBMED    32062022
REFERENCE   2  (bases 1 to 6460)
  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: "Protein 
            Expr Purif."; date: "2020-02-13"; volume: "170:105607. doi"; pages: 
            " 10.1016/j.pep.2020.105607"
COMMENT     Sequence Label: pMAK464
FEATURES             Location/Qualifiers
     source          1..6460
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(318..323,324..339,340..346)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    354..370
                     /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     359..381
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             539..877
                     /codon_start=1
                     /product="GFP-binding fragment of a single-chain camelid 
                     antibody (Rothbauer et al., 2008)"
                     /label=GFP nanobody
                     /translation="VQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKERE
                     WVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYW
                     GQGTQVTVS"
     CDS             932..949
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             959..982
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      994..1080
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1172..1199
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1227..1317
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1318..1386,1387..2178)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1536..1555)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2266..2854
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2755..2774
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3020..3039
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     protein_bind    3142..3166
                     /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             3181..3203
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3210..4172
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             3210..4172
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             4211..4228
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4281..4299)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4295..4342
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4472..4489
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     4672..4694
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4688..4879)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        5375..5452
                     /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."
     CDS             join(5453..6460,1..75)
                     /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(5472..5491)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
       61 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcacaatt
      121 ctcatgtttg acagcttatc atcgactgca cggtgcacca atgcttctgg cgtcaggcag
      181 ccatcggaag ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc
      241 aaggcgcact cccgttctgg ataatgtttt ttgcgccgac atcataacgg ttctggcaaa
      301 tattctgaaa tgagctgttg acaattaatc atcggctcgt ataatgtgtg gaattgtgag
      361 cggataacaa tttcacacag gaaacagcca gtccgtttag gtgttttcac gagcaattga
      421 ccaacaagga ccatagatta tgaaaataaa aacaggtgca cgcatcctcg cattatccgc
      481 attaacgacg atgatgtttt ccgcctcggc tctcgccaaa atcggatcca tggcgcaggt
      541 tcagctggtt gaaagcggtg gtgcactggt tcagcctggt ggtagcctgc gtctgagctg
      601 tgcagcaagc ggttttccgg ttaatcgtta tagcatgcgt tggtatcgtc aggcaccggg
      661 taaagaacgt gaatgggttg caggtatgag cagtgccggt gatcgtagca gctatgaaga
      721 tagcgttaaa ggtcgtttta ccatcagccg tgatgatgca cgtaataccg tttatctgca
      781 aatgaatagc ctgaaaccgg aagataccgc agtgtattat tgcaatgtta acgtgggctt
      841 tgaatattgg ggtcagggca cccaggttac cgttagcgat atcggactga acgacatctt
      901 cgaagctcag aaaatcgaat ggcacggctc ccatcaccat catcaccact aagaattcga
      961 ttacaaggat gacgacgata agtaaataag cttcaaataa aacgaaaggc tcagtcgaaa
     1021 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     1081 ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc
     1141 ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt
     1201 gcgtttctac aaactctttc ggtccgttgt ttatttttct aaatacattc aaatatgtat
     1261 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     1321 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     1381 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     1441 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     1501 gaacgtttcc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     1561 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     1621 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     1681 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     1741 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     1801 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     1861 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     1921 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     1981 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     2041 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     2101 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     2161 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgatttc
     2221 cttaggactg agcgtcaacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2281 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2341 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2401 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2461 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2521 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     2581 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     2641 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     2701 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     2761 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     2821 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     2881 gttcctggcc ttttgctggc cttttgctca catgttccgg taccgcgccc aacagtcccc
     2941 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     3001 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     3061 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa
     3121 attaatacga ctcactagag gggaattgtg agcggataac aattcccctc tagaaataat
     3181 tttgtttaac tttaagaagg agatatacca tgaaggataa caccgtgcca ctgaaattga
     3241 ttgccctgtt agcgaacggt gaatttcact ctggcgagca gttgggtgaa acgctgggaa
     3301 tgagccgggc ggctattaat aaacacattc agacactgcg tgactggggc gttgatgtct
     3361 ttaccgttcc gggtaaagga tacagcctgc ctgagcctat ccagttactt aatgctaaac
     3421 agatattggg tcagctggat ggcggtagtg tagccgtgct gccagtgatt gactccacga
     3481 atcagtacct tcttgatcgt atcggagagc ttaaatcggg cgatgcttgc attgcagaat
     3541 accagcaggc tggccgtggt cgccggggtc ggaaatggtt ttcgcctttt ggcgcaaact
     3601 tatatttgtc gatgttctgg cgtctggaac aaggcccggc ggcggcgatt ggtttaagtc
     3661 tggttatcgg tatcgtgatg gcggaagtat tacgcaagct gggtgcagat aaagttcgtg
     3721 ttaaatggcc taatgacctc tatctgcagg atcgcaagct ggcaggcatt ctggtggagc
     3781 tgactggcaa aactggcgat gcggcgcaaa tagtcattgg agccgggatc aacatggcaa
     3841 tgcgccgtgt tgaagagagt gtcgttaatc aggggtggat cacgctgcag gaagcgggga
     3901 tcaatctcga tcgtaatacg ttggcggcca tgctaatacg tgaattacgt gctgcgttgg
     3961 aactcttcga acaagaagga ttggcacctt atctgtcgcg ctgggaaaag ctggataatt
     4021 ttattaatcg cccagtgaaa cttatcattg gtgataaaga aatatttggc atttcacgcg
     4081 gaatagacaa acagggggct ttattacttg agcaggatgg aataataaaa ccctggatgg
     4141 gcggtgaaat atccctgcgt agtgcagaaa aaggtaagct ttgatgagga ctagtgcggc
     4201 cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga
     4261 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     4321 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatgct agcacatgtt
     4381 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4441 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     4501 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatataagg
     4561 tgcactgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     4621 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     4681 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     4741 catcagcgtg gtcgtgcagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     4801 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     4861 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     4921 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     4981 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     5041 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     5101 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     5161 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     5221 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     5281 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     5341 cccgtggcca ggacccaacg ctgcccgaaa ttccgacacc atcgaatggt gcaaaacctt
     5401 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     5461 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     5521 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     5581 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     5641 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     5701 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     5761 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     5821 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     5881 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     5941 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     6001 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     6061 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     6121 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     6181 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     6241 cgttggtgcg gatatttcgg tagtgggata cgacgatacc gaagacagct catgttatat
     6301 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     6361 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     6421 ggtgaaaaga aaaaccaccc tggcgcccaa tacacaaacc
//
