LOCUS       Exported                8759 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pD-Nus1, complete sequence.
ACCESSION   EF431918
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8759)
  AUTHORS   Ergin A, Bussow K, Sieper J, Thiel A, Duchmann R, Adam T.
  TITLE     Homologous High-Throughput Expression and Purification of Highly 
            Conserved E. coli Proteins
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8759)
  AUTHORS   Ergin A, Adam T, Bussow K.
  TITLE     Direct Submission
  JOURNAL   Submitted (09-FEB-2007) Gastroenterology, Charite Berlin (CBF), 
            Hindenburgdamm 30, Berlin 12200, Germany
REFERENCE   3  (bases 1 to 8759)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8759
                     /organism="Cloning vector pD-Nus1"
                     /mol_type="other DNA"
                     /db_xref="taxon:429339"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(582..1442)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=bla
                     /protein_id="ABO28856.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(582..1442)
                     /gene="bla"
                     /label=bla
     promoter        complement(1443..1535)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1616..2204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    2390..2532
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2634..2825)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3397..4479)
                     /codon_start=1
                     /gene="lacI"
                     /product="LacI"
                     /label=lacI
                     /note="Lac repressor"
                     /protein_id="ABO28857.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            complement(3397..4479)
                     /gene="lacI"
                     /label=lacI
     promoter        complement(4480..4557)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        4870..4888
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4870..4886
                     /regulatory_class="promoter"
                     /note="T7 promoter"
     protein_bind    4889..4913
                     /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             4928..4950
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     misc_feature    4970..4987
                     /label=His-tag
                     /note="His-tag"
     CDS             4970..4987
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    4994..6478
                     /label=Nus-tag
                     /note="Nus-tag"
     CDS             4994..6478
                     /codon_start=1
                     /gene="nusA"
                     /product="transcription elongation protein (N utilization 
                     substance protein A)"
                     /label=NusA
                     /note="highly soluble in E. coli"
                     /translation="MNKEILAVVEAVSNEKALPREKIFEALESALATATKKKYEQEIDV
                     RVQIDRKSGDFDTFRRWLVVDEVTQPTKEITLEAARYEDESLNLGDYVEDQIESVTFDR
                     ITTQTAKQVIVQKVREAERAMVVDQFREHEGEIITGVVKKVNRDNISLDLGNNAEAVIL
                     REDMLPRENFRPGDRVRGVLYSVRPEARGAQLFVTRSKPEMLIELFRIEVPEIGEEVIE
                     IKAAARDPGSRAKIAVKTNDKRIDPVGACVGMRGARVQAVSTELGGERIDIVLWDDNPA
                     QFVINAMAPADVASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRLASQLSGWELNVMT
                     VDDLQAKHQAEAHAAIDTFTKYLDIDEDFATVLVEEGFSTLEELAYVPMKELLEIEGLD
                     EPTVEALRERAKNALATIAQAQEESLGDNKPADDLLNLEGVDRDLAFKLAARGVCTLED
                     LAEQGIDDLADIEGLTDEKAGALIMAARNICWFGDEA"
     misc_feature    6494..6511
                     /label=His-tag
                     /note="His-tag"
     CDS             6494..6511
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    6515..6639
                     /label=attR1 recombination site
                     /note="attR1 recombination site"
     protein_bind    6518..6642
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        6667..6697
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             6751..7410
                     /codon_start=1
                     /gene="cmR"
                     /product="chloramphenicol resistance protein"
                     /label=cmR
                     /protein_id="ABO28854.1"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     gene            6751..7410
                     /gene="cmR"
                     /label=cmR
     CDS             7752..8057
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB"
                     /label=ccdB
                     /protein_id="ABO28855.1"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     gene            7752..8057
                     /gene="ccdB"
                     /label=ccdB
     protein_bind    complement(8098..8222)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    8101..8225
                     /label=attR2 recombination site
                     /note="attR2 recombination site"
     CDS             8231..8248
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      8414..8500
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      8592..8619
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      8687..8734
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"