LOCUS       Exported                6133 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Optimized bacterial expression plasmid of bdNEDP1 protease (Frey and
            Goerlich, 2014).
ACCESSION   .
VERSION     .
KEYWORDS    pDG02583
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6133)
  AUTHORS   Pleiner T,  Bates M,  Gorlich D
  TITLE     A toolbox of anti–mouse and anti–rabbit IgG secondary nanobodies
  JOURNAL   J Cell Biol Dec 2017, jcb.201709115
REFERENCE   2  (bases 1 to 6133)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6133
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     965..984
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1004..1081)
                     /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(1165..1187)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1273..1413
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1328..1347
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1428..1445)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1599..2187)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1679..1698)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2278..2296
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2320..2364
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    2340..2356
                     /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    2372..2388
                     /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             2407..2418
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2503..3600
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      3457..3466
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     primer_bind     3574..3597
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     terminator      4688..4782
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(4830..4849)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4841..5656
                     /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(4914..4933)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      5875..5961
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(6054..1003)
                     /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"
ORIGIN
        1 gcgtattggg ggccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
       61 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
      121 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
      181 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
      241 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
      301 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
      361 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
      421 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
      481 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
      541 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
      601 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
      661 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
      721 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
      781 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
      841 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
      901 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
      961 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     1021 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcaccat tcgatggtgt
     1081 cggaatttcg ggcagcgttg ggtcctggcc acgggtgcgc atgatctaga gctgcctcgc
     1141 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggcat ccgcttacag
     1201 acaagctgtg accgtctccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     1261 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt
     1321 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg
     1381 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     1441 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     1501 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     1561 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     1621 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     1681 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     1741 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     1801 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     1861 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     1921 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     1981 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     2041 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     2101 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     2161 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     2221 gctcagtgga acgaaaactc acgttaaggg attttggtca tgacattaac ctataaaaat
     2281 aggcgtatca cgaggccctt tcgtcttcac ctcgagaaat cataaaaaat ttatttgctt
     2341 tgtgagcgga taacaattat aatagattca attgtgagcg gataacaatt tcacacagaa
     2401 ttcattaaag aggagaaatt aaccatgagc aagcatcacc atcattcagg ccatcaccat
     2461 accggacacc accatcattc aggcagtcat caccataccg ggaaaactga agaaggtaaa
     2521 ctggtaatct ggattaacgg cgataaaggc tataacggtc tcgctgaagt cggtaagaaa
     2581 ttcgagaaag ataccggaat taaagtcacc gttgagcatc cggataaact ggaagagaaa
     2641 ttcccacagg ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc
     2701 tttggtggct acgctcaatc tggcctgttg gctgaaatca ccccggacaa agcgttccag
     2761 gacaagctgt atccgtttac ctgggatgcc gtacgttaca acggcaagct gattgcttac
     2821 ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag atctgctgcc gaacccgcca
     2881 aaaacctggg aagagatccc ggcgctggat aaagaactga aagcgaaagg taagagcgcg
     2941 ctgatgttca acctgcaaga accgtacttc acctggccgc tgattgctgc tgacgggggt
     3001 tatgcgttca agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct
     3061 ggcgcgaaag cgggtctgac cttcctggtt gacctgatta aaaacaaaca catgaatgca
     3121 gacactgatt actccatcgc agaagctgcc tttaataaag gcgaaacagc gatgaccatc
     3181 aacggcccgt gggcatggtc caacatcgac accagcaaag tgaattatgg tgtaacggta
     3241 ctgccgacct tcaagggtca accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt
     3301 aacgccgcca gtccgaacaa agagctggca aaagagttcc tcgaaaacta tctgctgact
     3361 gatgaaggtc tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct
     3421 tacgaggaag agttggcgaa agatccacgt attgccgcca ccatggaaaa cgcccagaaa
     3481 ggtgaaatca tgccgaacat cccgcagatg tccgctttct ggtatgccgt gcgtactgcg
     3541 gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact
     3601 aattcgagct cagctgcggg tggcgaagaa gataagaaac cggcaggtgg cgaaggtggc
     3661 ggtgcccata tcaacctgaa agtgaaaggt caagacggca acgaagtctt tttccgcatc
     3721 aaacgttcta cccagctgaa aaagctgatg aacgcatact gtgaccgtca gtctgtagac
     3781 atgaccgcaa ttgctttcct gtttgatggt cgtcgcctgc gtgcggaaca gaccccggat
     3841 gaactggaga tggaagatgg cgacgaaatc gacgcaatgc ttcatcagac tggtggcggt
     3901 ggaatggatg aacgtgtcct gtcttatggt gatgtggtgc tccttcgttc cgatctggct
     3961 attctgcgtg gcccgcactt cctgaacgac cgtattatcg ccttctatct ggcacacctg
     4021 tccgcttcct tccacggtga tggcgatctg ctgctcctgc cgccgagcat tccgtacctg
     4081 ctgtccaacc tgccggaccc ggaatctgtc gcagaaccac tgtgtctggc atcccgccgt
     4141 ctggtcctgc tgccggttaa cgataacccg gacgcctcgg tggcaaatgg cggttctcac
     4201 tggactctgc tcgtcctgga cgctgcgact actgacccgc aggctccgcg tttcgtgcat
     4261 catgactccc tgcgcggttc cgcgaacgcg gctgctgcgc gtcgcctggc ccgtgctctg
     4321 accgctggtg gtgcgccgct gcgcttcgtt gaggctccta ccccgaccca gcgtaacggc
     4381 cacgactgcg gcgtctatgt cctggccgta gctcgtgcta tttgtggttg gtggcgttct
     4441 tctcgtcgcc gcgaaaacca gcagggcggt ggcggcgatt ggtttgcaac catgatggaa
     4501 gaagtggatg ctgaatccgt aggcgctatg cgcgctgaac tgctccagct cattcaccgt
     4561 ctgattcagg acaaggaaca agaagaagag aaaaagtcca aagctggcgt ggaggatacc
     4621 tgcggccaat aactcgtagg ctcgacacga agcatagcgg ccgcataagc ttaattagct
     4681 gagcttggac tcctgttgat agatccagta atgacctcag aactccatct ggatttgttc
     4741 agaacgctcg gttgccgccg ggcgtttttt attggtgaga atccaagcta gccatgaaaa
     4801 taaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga
     4861 aacgtcttgc tctaggccgc gattaaattc caacatggat gctgatttat atgggtataa
     4921 atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc
     4981 cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga
     5041 tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt
     5101 tatccgtact cctgatgatg cttggttact cacgactgcg atccccggca aaacagcatt
     5161 ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt
     5221 cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt
     5281 tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga
     5341 tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcaca aacttttgcc
     5401 attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga
     5461 cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca
     5521 ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct
     5581 ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct
     5641 cgatgagttt ttctaagaat taattcatgg gcaaatatta tacgcaaggc gacaaggtgc
     5701 tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa
     5761 tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa tttttttaag
     5821 gcagttattg gtgcccttaa acgcctgggg taatgactct ctagcttgag gcatcaaata
     5881 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     5941 gctctcctga gtaggacaaa tccgccctct agattacgtg cagtcgatga taagctgtca
     6001 aacatgagaa ttgtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     6061 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     6121 ggagaggcgg ttt
//
