LOCUS       sequence-298981-        6466 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NB-alpha-Rb-IgG-Fc.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6466)
  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 6466)
  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: pMAK461
FEATURES             Location/Qualifiers
     source          1..6466
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(720..725,726..741,742..748)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    756..772
                     /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     761..783
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1340..1357
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1367..1390
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      1402..1488
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1580..1607
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1635..1725
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1726..1794,1795..2586)
                     /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(1944..1963)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2674..3262
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3163..3182
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3428..3447
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     protein_bind    3550..3574
                     /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             3589..3611
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3618..4580
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             3618..4580
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             4619..4636
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4689..4707)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4703..4750
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4880..4897
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5080..5102
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5096..5287)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        5783..5860
                     /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(5861..6466,1..477)
                     /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(5880..5899)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc
       61 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg
      121 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg
      181 cgcgttggtg cggatatttc ggtagtggga tacgacgata ccgaagacag ctcatgttat
      241 atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac
      301 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca
      361 ctggtgaaaa gaaaaaccac cctggcgccc aatacacaaa ccgcctctcc ccgcgcgttg
      421 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
      481 caacgcaatt aatgtaagtt agctcactca ttaggcacaa ttctcatgtt tgacagctta
      541 tcatcgactg cacggtgcac caatgcttct ggcgtcaggc agccatcgga agctgtggta
      601 tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct
      661 ggataatgtt ttttgcgccg acatcataac ggttctggca aatattctga aatgagctgt
      721 tgacaattaa tcatcggctc gtataatgtg tggaattgtg agcggataac aatttcacac
      781 aggaaacagc cagtccgttt aggtgttttc acgagcaatt gaccaacaag gaccatagat
      841 tatgaaaata aaaacaggtg cacgcatcct cgcattatcc gcattaacga cgatgatgtt
      901 ttccgcctcg gctctcgcca aaatcggatc ccaggtgcaa ttggtagagt ctgggggagg
      961 cttggtgcag gctggtgact ctctgcgact ctcctgtgta gcctctgggc gctccttgga
     1021 tggggccacc atgcgctggt accgccaggc tccagggaag gagcgagaat ttgttgcagg
     1081 cattttttgg gatgagattg gtacggaata tgcagacact gcgaagggcc gattcaccat
     1141 ctccagagac aatgccaaga acacgatata tctacaaatg accaacctga ggtccgaaga
     1201 cacggccatg tattattgta atggattagt attcgggggt gaatactggg gacaggggac
     1261 ccaggtcaca gtctcctccg gtggagatat cggactgaac gacatcttcg aagctcagaa
     1321 aatcgaatgg cacggctccc atcaccatca tcaccactaa gaattcgatt acaaggatga
     1381 cgacgataag taaataagct tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
     1441 cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg
     1501 gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact
     1561 gccaggcatc aaattaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa
     1621 actctttcgg tccgttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     1681 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     1741 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     1801 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     1861 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgtttccca
     1921 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
     1981 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     2041 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     2101 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     2161 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     2221 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     2281 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     2341 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     2401 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     2461 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     2521 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     2581 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttcct taggactgag
     2641 cgtcaacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     2701 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     2761 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     2821 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     2881 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     2941 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     3001 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     3061 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     3121 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     3181 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     3241 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     3301 ttgctggcct tttgctcaca tgttccggta ccgcgcccaa cagtcccccg gccacggggc
     3361 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     3421 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     3481 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact
     3541 cactagaggg gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt
     3601 taagaaggag atataccatg aaggataaca ccgtgccact gaaattgatt gccctgttag
     3661 cgaacggtga atttcactct ggcgagcagt tgggtgaaac gctgggaatg agccgggcgg
     3721 ctattaataa acacattcag acactgcgtg actggggcgt tgatgtcttt accgttccgg
     3781 gtaaaggata cagcctgcct gagcctatcc agttacttaa tgctaaacag atattgggtc
     3841 agctggatgg cggtagtgta gccgtgctgc cagtgattga ctccacgaat cagtaccttc
     3901 ttgatcgtat cggagagctt aaatcgggcg atgcttgcat tgcagaatac cagcaggctg
     3961 gccgtggtcg ccggggtcgg aaatggtttt cgccttttgg cgcaaactta tatttgtcga
     4021 tgttctggcg tctggaacaa ggcccggcgg cggcgattgg tttaagtctg gttatcggta
     4081 tcgtgatggc ggaagtatta cgcaagctgg gtgcagataa agttcgtgtt aaatggccta
     4141 atgacctcta tctgcaggat cgcaagctgg caggcattct ggtggagctg actggcaaaa
     4201 ctggcgatgc ggcgcaaata gtcattggag ccgggatcaa catggcaatg cgccgtgttg
     4261 aagagagtgt cgttaatcag gggtggatca cgctgcagga agcggggatc aatctcgatc
     4321 gtaatacgtt ggcggccatg ctaatacgtg aattacgtgc tgcgttggaa ctcttcgaac
     4381 aagaaggatt ggcaccttat ctgtcgcgct gggaaaagct ggataatttt attaatcgcc
     4441 cagtgaaact tatcattggt gataaagaaa tatttggcat ttcacgcgga atagacaaac
     4501 agggggcttt attacttgag caggatggaa taataaaacc ctggatgggc ggtgaaatat
     4561 ccctgcgtag tgcagaaaaa ggtaagcttt gatgaggact agtgcggccg cactcgagca
     4621 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     4681 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     4741 gggttttttg ctgaaaggag gaactatatc cggatgctag cacatgttct ttcctgcgtt
     4801 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     4861 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     4921 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atataaggtg cactgtgact
     4981 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     5041 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     5101 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     5161 cgtgcagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     5221 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     5281 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     5341 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     5401 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     5461 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     5521 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     5581 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     5641 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     5701 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg
     5761 acccaacgct gcccgaaatt ccgacaccat cgaatggtgc aaaacctttc gcggtatggc
     5821 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
     5881 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     5941 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     6001 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     6061 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     6121 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     6181 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     6241 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     6301 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     6361 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     6421 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcat
//
