LOCUS       sequence-298982-        6478 bp    DNA     circular SYN 14-APR-2025
DEFINITION  NB-alpha-Ms/Rt-IgG1-Fc.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6478)
  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 6478)
  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: "Protein 
            Expr Purif."; date: "2020-02-13"; volume: "170:105607. doi"; pages: 
            " 10.1016/j.pep.2020.105607"
COMMENT     Sequence Label: pMAK462
FEATURES             Location/Qualifiers
     source          1..6478
                     /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             950..967
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             977..1000
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      1012..1098
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1190..1217
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1245..1335
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1336..1404,1405..2196)
                     /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(1554..1573)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2284..2872
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2773..2792
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3038..3057
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     protein_bind    3160..3184
                     /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             3199..3221
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3228..4190
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             3228..4190
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             4229..4246
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4299..4317)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4313..4360
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4490..4507
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     4690..4712
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4706..4897)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        5393..5470
                     /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(5471..6478,1..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(5490..5509)
                     /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 tggtgcagcc tggggggtct ctgaggctct cctgtgcagc
      601 ctctggattc acattcagtg atacctggat gaactgggtc cgccaggctc cagggaaggg
      661 gctctattgg atctcagcca tcaatccgga cggtggtaac acagcctatg cagactccgt
      721 caagggccgg ttcaccatct ccagagacaa cgccaagaac atggtgtatc tgcagatgga
      781 caacctgaga cctgaggaca cggccatgta ttactgcgca aagggttggg ttcgccttcc
      841 agatcccgat ctggtaaggg gccaggggac ccaggtcaca gtctcctccg gtggagatat
      901 cggactgaac gacatcttcg aagctcagaa aatcgaatgg cacggctccc atcaccatca
      961 tcaccactaa gaattcgatt acaaggatga cgacgataag taaataagct tcaaataaaa
     1021 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
     1081 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga
     1141 gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca gaaggccatc
     1201 ctgacggatg gcctttttgc gtttctacaa actctttcgg tccgttgttt atttttctaa
     1261 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     1321 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     1381 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     1441 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     1501 gagagttttc gccccgaaga acgtttccca atgatgagca cttttaaagt tctgctatgt
     1561 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
     1621 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     1681 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     1741 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     1801 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     1861 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     1921 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     1981 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     2041 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     2101 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     2161 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     2221 atactttaga ttgatttcct taggactgag cgtcaacccc gtagaaaaga tcaaaggatc
     2281 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     2341 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     2401 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     2461 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     2521 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     2581 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     2641 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     2701 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     2761 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     2821 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     2881 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttccggta
     2941 ccgcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     3001 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     3061 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     3121 agatctcgat cccgcgaaat taatacgact cactagaggg gaattgtgag cggataacaa
     3181 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg aaggataaca
     3241 ccgtgccact gaaattgatt gccctgttag cgaacggtga atttcactct ggcgagcagt
     3301 tgggtgaaac gctgggaatg agccgggcgg ctattaataa acacattcag acactgcgtg
     3361 actggggcgt tgatgtcttt accgttccgg gtaaaggata cagcctgcct gagcctatcc
     3421 agttacttaa tgctaaacag atattgggtc agctggatgg cggtagtgta gccgtgctgc
     3481 cagtgattga ctccacgaat cagtaccttc ttgatcgtat cggagagctt aaatcgggcg
     3541 atgcttgcat tgcagaatac cagcaggctg gccgtggtcg ccggggtcgg aaatggtttt
     3601 cgccttttgg cgcaaactta tatttgtcga tgttctggcg tctggaacaa ggcccggcgg
     3661 cggcgattgg tttaagtctg gttatcggta tcgtgatggc ggaagtatta cgcaagctgg
     3721 gtgcagataa agttcgtgtt aaatggccta atgacctcta tctgcaggat cgcaagctgg
     3781 caggcattct ggtggagctg actggcaaaa ctggcgatgc ggcgcaaata gtcattggag
     3841 ccgggatcaa catggcaatg cgccgtgttg aagagagtgt cgttaatcag gggtggatca
     3901 cgctgcagga agcggggatc aatctcgatc gtaatacgtt ggcggccatg ctaatacgtg
     3961 aattacgtgc tgcgttggaa ctcttcgaac aagaaggatt ggcaccttat ctgtcgcgct
     4021 gggaaaagct ggataatttt attaatcgcc cagtgaaact tatcattggt gataaagaaa
     4081 tatttggcat ttcacgcgga atagacaaac agggggcttt attacttgag caggatggaa
     4141 taataaaacc ctggatgggc ggtgaaatat ccctgcgtag tgcagaaaaa ggtaagcttt
     4201 gatgaggact agtgcggccg cactcgagca ccaccaccac caccactgag atccggctgc
     4261 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     4321 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     4381 cggatgctag cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     4441 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     4501 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     4561 ttcacaccgc atataaggtg cactgtgact gggtcatggc tgcgccccga cacccgccaa
     4621 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4681 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4741 ggcagctgcg gtaaagctca tcagcgtggt cgtgcagcga ttcacagatg tctgcctgtt
     4801 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     4861 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     4921 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     4981 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     5041 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     5101 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     5161 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     5221 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     5281 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     5341 ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgaaatt ccgacaccat
     5401 cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag
     5461 ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta
     5521 tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa
     5581 agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc
     5641 gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc
     5701 gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc
     5761 gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca
     5821 acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga
     5881 agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa
     5941 cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt
     6001 gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg
     6061 tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga
     6121 aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat
     6181 cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat
     6241 taccgagtcc gggctgcgcg ttggtgcgga tatttcggta gtgggatacg acgataccga
     6301 agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg
     6361 gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca
     6421 gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cacaaacc
//
