LOCUS       sequence-298983-        6496 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NB-alpha-Ms-IgK.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6496)
  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 6496)
  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: pMAK463
FEATURES             Location/Qualifiers
     source          1..6496
                     /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             968..985
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             995..1018
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      1030..1116
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1208..1235
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1263..1353
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1354..1422,1423..2214)
                     /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(1572..1591)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2302..2890
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2791..2810
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3056..3075
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     protein_bind    3178..3202
                     /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             3217..3239
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3246..4208
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             3246..4208
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             4247..4264
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4317..4335)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4331..4378
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4508..4525
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     4708..4730
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4724..4915)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        5411..5488
                     /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(5489..6496,1..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(5508..5527)
                     /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 atcggatccc aggtgcaatt
      541 ggtggagtct gggggaggct gggtgcagcc tggggggtct ctgagactct cctgtgcagc
      601 ctctggattc accttcagtg acactgccat gatgtgggtc cgccaggctc cagggaaggg
      661 gcgagagtgg gtcgcagcta ttgatactgg tggtggatac acatactatg cagactccgt
      721 gaagggccga ttcaccatct ccagagacaa cgccaagaac acgctgtatc tgcaaatgaa
      781 cagcctgaaa ccggaggaca cggcccgata ctactgtgcg aagacctact ccggtaatta
      841 ctatagcaac tacacggttg ccaactatgg caccacgggc cgggggaccc tggtcacagt
      901 ctcctccggt ggagatatcg gactgaacga catcttcgaa gctcagaaaa tcgaatggca
      961 cggctcccat caccatcatc accactaaga attcgattac aaggatgacg acgataagta
     1021 aataagcttc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
     1081 tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt
     1141 gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa
     1201 attaagcaga aggccatcct gacggatggc ctttttgcgt ttctacaaac tctttcggtc
     1261 cgttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     1321 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     1381 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     1441 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     1501 aacagcggta agatccttga gagttttcgc cccgaagaac gtttcccaat gatgagcact
     1561 tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc
     1621 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     1681 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     1741 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     1801 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     1861 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     1921 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     1981 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     2041 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     2101 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     2161 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     2221 gaccaagttt actcatatat actttagatt gatttcctta ggactgagcg tcaaccccgt
     2281 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     2341 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     2401 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     2461 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     2521 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     2581 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     2641 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     2701 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     2761 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     2821 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     2881 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     2941 tgctcacatg ttccggtacc gcgcccaaca gtcccccggc cacggggcct gccaccatac
     3001 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     3061 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     3121 gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca ctagagggga
     3181 attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta agaaggagat
     3241 ataccatgaa ggataacacc gtgccactga aattgattgc cctgttagcg aacggtgaat
     3301 ttcactctgg cgagcagttg ggtgaaacgc tgggaatgag ccgggcggct attaataaac
     3361 acattcagac actgcgtgac tggggcgttg atgtctttac cgttccgggt aaaggataca
     3421 gcctgcctga gcctatccag ttacttaatg ctaaacagat attgggtcag ctggatggcg
     3481 gtagtgtagc cgtgctgcca gtgattgact ccacgaatca gtaccttctt gatcgtatcg
     3541 gagagcttaa atcgggcgat gcttgcattg cagaatacca gcaggctggc cgtggtcgcc
     3601 ggggtcggaa atggttttcg ccttttggcg caaacttata tttgtcgatg ttctggcgtc
     3661 tggaacaagg cccggcggcg gcgattggtt taagtctggt tatcggtatc gtgatggcgg
     3721 aagtattacg caagctgggt gcagataaag ttcgtgttaa atggcctaat gacctctatc
     3781 tgcaggatcg caagctggca ggcattctgg tggagctgac tggcaaaact ggcgatgcgg
     3841 cgcaaatagt cattggagcc gggatcaaca tggcaatgcg ccgtgttgaa gagagtgtcg
     3901 ttaatcaggg gtggatcacg ctgcaggaag cggggatcaa tctcgatcgt aatacgttgg
     3961 cggccatgct aatacgtgaa ttacgtgctg cgttggaact cttcgaacaa gaaggattgg
     4021 caccttatct gtcgcgctgg gaaaagctgg ataattttat taatcgccca gtgaaactta
     4081 tcattggtga taaagaaata tttggcattt cacgcggaat agacaaacag ggggctttat
     4141 tacttgagca ggatggaata ataaaaccct ggatgggcgg tgaaatatcc ctgcgtagtg
     4201 cagaaaaagg taagctttga tgaggactag tgcggccgca ctcgagcacc accaccacca
     4261 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     4321 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     4381 gaaaggagga actatatccg gatgctagca catgttcttt cctgcgttat cccctgattc
     4441 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     4501 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     4561 tacgcatctg tgcggtattt cacaccgcat ataaggtgca ctgtgactgg gtcatggctg
     4621 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     4681 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     4741 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgcagcgatt
     4801 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     4861 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     4921 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     4981 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     5041 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     5101 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     5161 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     5221 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     5281 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     5341 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggccaggac ccaacgctgc
     5401 ccgaaattcc gacaccatcg aatggtgcaa aacctttcgc ggtatggcat gatagcgccc
     5461 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
     5521 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
     5581 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     5641 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     5701 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     5761 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     5821 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     5881 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     5941 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     6001 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     6061 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     6121 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     6181 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     6241 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tttcggtagt
     6301 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     6361 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     6421 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     6481 gcccaataca caaacc
//
