LOCUS       40924_17996        7295 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector for ligation-independent cloning (LIC) to express 
            NusA-tagged proteins with an enterokinase site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7295)
  AUTHORS   Novagen (EMD Millipore)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 7295)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     For LIC, linearize with BseRI and treat with T4 DNA polymerase plus 
            dTTP.
FEATURES             Location/Qualifiers
     source          1..7295
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      complement(141..168)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(260..346)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     CDS             complement(512..529)
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             complement(536..568)
                     /codon_start=1
                     /label=HSV tag
                     /note="HSV (herpes simplex virus) epitope tag"
                     /translation="QPELAPEDPED"
     CDS             complement(701..715)
                     /codon_start=1
                     /label=enterokinase site
                     /note="enterokinase recognition and cleavage site"
                     /translation="DDDDK"
     CDS             complement(734..751)
                     /codon_start=1
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
                     /translation="LVPRGS"
     CDS             complement(767..811)
                     /codon_start=1
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(821..838)
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             complement(854..2338)
                     /codon_start=1
                     /label=NusA
                     /note="transcription elongation protein (N utilization
                     substance protein A)"
                     /translation="MNKEILAVVEAVSNEKALPREKIFEALESALATATKKKYEQEIDV
                     RVQIDRKSGDFDTFRRWLVVDEVTQPTKEITLEAARYEDESLNLGDYVEDQIESVTFDR
                     ITTQTAKQVIVQKVREAERAMVVDQFREHEGEIITGVVKKVNRDNISLDLGNNAEAVIL
                     REDMLPRENFRPGDRVRGVLYSVRPEARGAQLFVTRSKPEMLIELFRIEVPEIGEEVIE
                     IKAAARDPGSRAKIAVKTNDKRIDPVGACVGMRGARVQAVSTELGGERIDIVLWDDNPA
                     QFVINAMAPADVASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRLASQLSGWELNVMT
                     VDDLQAKHQAEAHAAIDTFTKYLDIDEDFATVLVEEGFSTLEELAYVPMKELLEIEGLD
                     EPTVEALRERAKNALATIAQAQEESLGDNKPADDLLNLEGVDRDLAFKLAARGVCTLED
                     LAEQGIDDLADIEGLTDEKAGALIMAARNICWFGDEA"
     RBS             complement(2346..2368)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(2383..2407)
                     /label=lac operator
                     /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)."
     promoter        complement(2408..2426)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2739..2816
                     /label=lacI promoter
     CDS             2817..3896
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    3912..3933
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             4471..4659
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    4764..4906
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(5092..5680)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        5761..5853
                     /label=AmpR promoter
     CDS             5854..6711
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      complement(6829..7284)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggata catatttgaa tgtatttaga aaaataaaca
      121 aaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc ttaatttgat
      181 gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc ttcgcaacgt
      241 tcaaatccgc tcccggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga
      301 taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctggcagttc
      361 cctactctcg catggggaga ccccacacta ccatcggcgc tacggcgttt cacttctgag
      421 ttcggcatgg ggtcaggtgg gaccaccgcg ctactgccgc caggcaaatt ctgttttatc
      481 agcctaggaa cgcccaactt aattaacatt agtggtggtg gtggtggtgc tcgagatcct
      541 ctgggtcttc aggagcgagt tctggctggc ttgcacgtgt atacagctgt gcggccgcaa
      601 gcttcgaacg cgtatcgatg gtaccgtcga cgtcctgcag gcgcgcctgt acagaattcg
      661 gatccacgag ctcagatatc gttgaggaga agcccggact cttgtcgtcg tcatcaatcg
      721 taccagaacc cgcgctgccc cgggggacca gaccagttgg cggtggcgag tccatgtgct
      781 ggcgttcaaa tttcgcagca gcggtttctt tacccgcgga gtgatggtga tggtgatgac
      841 cagaaccact agtcgcttcg tcaccgaacc agcaaatatt acgggcagcc ataatcagtg
      901 ctccggcttt ttcgtcggtc aacccttcga tatcagccag atcatcaatg ccctgttcgg
      961 cgagatcttc cagcgtacaa acgccacggg cggccagttt gaatgccaaa tcacgatcta
     1021 ccccttcaag gttcagcaga tcgtcagccg gtttgttatc accgaggctt tcttcctggg
     1081 cctgtgcaat ggtggccagt gcatttttag cacgctcgcg cagtgcttca acggtcggct
     1141 catcaaggcc ttcgatttcc aacagctctt tcatcggcac ataggccaat tcttccagcg
     1201 tcgagaagcc ttcttctacc agaacagtcg cgaagtcttc gtcgatgtcg agatatttgg
     1261 tgaaggtgtc gatcgctgcg tgcgcttccg cctgatgctt agcttgcagg tcgtcaacgg
     1321 tcatcacgtt gagttcccaa ccgctcagtt gcgaagccag acgcacgttc tgaccgttac
     1381 ggccaatcgc ctgcgccaga ttaccggctt caacggcgat gtccatggtg tgtttatctt
     1441 catccaccac gatagaagca acgtctgccg gtgccattgc gttaatcacg aactgcgccg
     1501 ggttatcatc ccacaggacg atatcgatac gctcgccacc cagttcagta gacaccgcct
     1561 gaacacgcgc gccacgcata cctacgcaag cacctaccgg atcgatacgt ttatcgttgg
     1621 ttttcaccgc gattttcgca cgagaacccg gatcgcgagc cgctgcttta atttcaatca
     1681 cttcttcgcc gatttctggc acttcaatac ggaacagttc gatcagcatt tccggcttgg
     1741 aacgagtgac gaacagttgc gcgccacgcg cttccgggcg aacggaatag agcacgccac
     1801 gaacgcggtc gccagggcgg aagttttcac gcggcagcat atcttcgcgc aggatcacgg
     1861 cttcagcgtt gttgcccaga tccagagaga tgttgtcgcg gtttactttt ttcaccacgc
     1921 cggtgatgat ttcaccttcg tgttcacgga actgatcaac caccatcgca cgttcggctt
     1981 cacgcacttt ctgcacgata acctgttttg ccgtctgggt agtgatacgg tcaaaggtaa
     2041 cagactcaat ctgatcttca acgtaatcgc ccaggttcag gctttcatct tcataacgtg
     2101 cggcttcaag ggtgatttcc ttggtcggct gggtgacttc atcaacaact aaccagcgac
     2161 ggaaagtgtc aaaatcaccg cttttgcgat cgatctgtac gcggacgtcg atctcttgtt
     2221 catatttttt ctttgttgct gtcgccagcg cgctttccaa tgcttcgaaa atcttctcgc
     2281 gaggtagcgc cttttcattg gatacggctt caactacagc caaaatttct ttgttcatat
     2341 gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca
     2401 caattcccct atagtgagtc gtattaattt cgcgggatcg agatcgatct cgatcctcta
     2461 cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat
     2521 cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt
     2581 cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca
     2641 tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct
     2701 aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa
     2761 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
     2821 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     2881 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     2941 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     3001 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     3061 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     3121 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     3181 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     3241 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     3301 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     3361 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     3421 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     3481 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     3541 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     3601 tgcgcgttgg tgcggacatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     3661 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     3721 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     3781 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     3841 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     3901 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc
     3961 ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac tagcatgatc
     4021 gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat
     4081 gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga
     4141 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca
     4201 gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga
     4261 acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc
     4321 cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca
     4381 tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg
     4441 aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac
     4501 atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac
     4561 gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc
     4621 agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     4681 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     4741 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     4801 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac catatatgcg
     4861 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc
     4921 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4981 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5041 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5101 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5161 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5221 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5281 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5341 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5401 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5461 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5521 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5581 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5641 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5701 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgaccta
     5761 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5821 aaatgcttca atcatgattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5881 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     5941 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     6001 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     6061 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     6121 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     6181 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     6241 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     6301 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     6361 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     6421 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     6481 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     6541 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     6601 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     6661 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     6721 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6781 atctaggtga agatcctttt tgataatctc atgccatcgt gccgccagaa attgtaaacg
     6841 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     6901 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     6961 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     7021 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     7081 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     7141 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     7201 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     7261 ttaatgcgcc gctacagggc gcgtcccatt cgcca
//
