LOCUS       pNYCOMPS-LIC-FH1        6858 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector for high-throughput expression and purification of 
            membrane proteins with N-terminal FLAG(R) and 10xHis tags.
ACCESSION   .
VERSION     .
KEYWORDS    pNYCOMPS-LIC-FH10T+ (N term).
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6858)
  AUTHORS   Mancia F, Love J.
  TITLE     High-throughput expression and purification of membrane proteins.
  JOURNAL   J. Struct. Biol. 2010;172:85-93.
  PUBMED    20394823
REFERENCE   2  (bases 1 to 6858)
  AUTHORS   New York Structural Biology Center
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6858)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J. Struct. 
            Biol."; date: "2010"; volume: "172"; pages: "85-93"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     For ligation-independent cloning (LIC), linearize with SnaBI and 
            treat with T4 DNA polymerase plus dGTP.
FEATURES             Location/Qualifiers
     source          1..6858
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             6..23
                     /label=6xHis
                     /note="6xHis affinity tag"
     terminator      90..137
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      174..629
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(725..1537)
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     rep_origin      1659..2247
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(2433..2575)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2680..2868)
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     protein_bind    complement(3643..3664)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3680..4759)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4760..4837)
                     /label=lacI promoter
     promoter        5146..5164
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5165..5189
                     /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)."
     RBS             5204..5226
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5233..5235
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             5236..5259
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             5260..5286
                     /label=9xHis
                     /note="9xHis affinity tag"
     CDS             5290..5310
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(5393..5695)
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
     CDS             complement(6040..6696)
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     promoter        complement(6750..6780)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
ORIGIN
        1 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
       61 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
      121 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
      181 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
      241 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
      301 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
      361 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
      421 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
      481 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
      541 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
      601 ttttaacaaa atattaacgt ttacaatttc aggtggcact tttcggggaa atgtgcgcgg
      661 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
      721 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
      781 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
      841 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
      901 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
      961 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     1021 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     1081 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     1141 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     1201 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     1261 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     1321 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     1381 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     1441 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     1501 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     1561 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     1621 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     1681 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     1741 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     1801 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     1861 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     1921 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     1981 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     2041 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2101 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2161 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     2221 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2281 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     2341 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     2401 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     2461 ctgtgcggta tttcacaccg catatatggt gcactctcag tacaatctgc tctgatgccg
     2521 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     2581 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     2641 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     2701 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     2761 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     2821 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     2881 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     2941 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     3001 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     3061 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     3121 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     3181 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     3241 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     3301 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     3361 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     3421 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     3481 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     3541 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     3601 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     3661 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     3721 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     3781 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     3841 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     3901 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     3961 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     4021 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     4081 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     4141 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     4201 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     4261 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     4321 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     4381 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     4441 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     4501 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     4561 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     4621 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     4681 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     4741 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     4801 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     4861 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     4921 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     4981 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     5041 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     5101 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta
     5161 taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga
     5221 aggagatata ccatggatta taaagatgat gatgataaac atcatcacca tcaccatcac
     5281 caccatcacg aaaacctgta ttttcaatcc tacgtaatac tgtaaaacac aacatatcca
     5341 gtcactatgg tcgacctgca gactggctgt gtataaggga gcctgacatt tatattcccc
     5401 agaacatcag gttaatggcg tttttgatgt cattttcgcg gtggctgaga tcagccactt
     5461 cttccccgat aacggagacc ggcacactgg ccatatcggt ggtcatcatg cgccagcttt
     5521 catccccgat atgcaccacc gggtaaagtt cacgggagac tttatctgac agcagacgtg
     5581 cactggccag ggggatcacc atccgtcgcc cgggcgtgtc aataatatca ctctgtacat
     5641 ccacaaacag acgataacgg ctctctcttt tataggtgta aaccttaaac tgcatttcac
     5701 cagtccctgt tctcgtcagc aaaagagccg ttcatttcaa taaaccgggc gacctcagcc
     5761 atcccttcct gattttccgc tttccagcgt tcggcacgca gacgacgggc ttcattctgc
     5821 atggttgtgc ttaccagacc ggagatattg acatcatata tgccttgagc aactgatagc
     5881 tgtcgctgtc aactgtcact gtaatacgct gcttcatagc acacctcttt ttgacatact
     5941 tcgggtatac atatcagtat atattcttat accgcaaaaa tcagcgcgca aatacgcata
     6001 ctgttatctg gcttttagta agccggatcc agatctttac gccccgccct gccactcatc
     6061 gcagtactgt tgtaattcat taagcattct gccgacatgg aagccatcac agacggcatg
     6121 atgaacctga atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat
     6181 ggtgaaaacg ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa
     6241 actcacccag ggattggctg agacgaaaaa catattctca ataaaccctt tagggaaata
     6301 ggccaggttt tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa actgccggaa
     6361 atcgtcgtgg tattcactcc agagcgatga aaacgtttca gtttgctcat ggaaaacggt
     6421 gtaacaaggg tgaacactat cccatatcac cagctcaccg tctttcattg ccatacggaa
     6481 ttccggatga gcattcatca ggcgggcaag aatgtgaata aaggccggat aaaacttgtg
     6541 cttatttttc tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg tctggttata
     6601 ggtacattga gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat
     6661 atcaacggtg gtatatccag tgattttttt ctccatttta gcttccttag ctcctgaaaa
     6721 tctcgacgga tcctaactca aaatccacac attatacgag ccggaagcat aaagtgtaaa
     6781 gcctggggtg cctaatgcgg ccgccatagt gactggatat gttgtgtttt acagtattac
     6841 gtataatatt gagggaac
//
