LOCUS       40924_8636        5825 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pCALnFLAG, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5825)
  AUTHORS   Felts K, Grafsky AJ.
  TITLE     pCALnFLAG
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5825)
  AUTHORS   Felts K, Grafsky AJ.
  TITLE     Direct Submission
  JOURNAL   Submitted (26-AUG-1998) Technical Services, Stratagene, 11011 N. 
            Torrey Pines Rd., La Jolla, CA 92037, USA
REFERENCE   3  (bases 1 to 5825)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5825)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (26-AUG-1998) Technical Services, Stratagene, 11011 N. Torrey Pines 
            Rd., La Jolla, CA 92037, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5825
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        10..38
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     terminator      complement(213..260)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(386..409)
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     CDS             complement(410..427)
                     /codon_start=1
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
                     /translation="LVPRGS"
     CDS             complement(428..505)
                     /codon_start=1
                     /label=CBP
                     /note="calmodulin-binding peptide"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     RBS             complement(516..538)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(553..577)
                     /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(578..596)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        905..982
                     /label=lacI promoter
     CDS             983..2062
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    2078..2099
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3377..3565
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    3670..3812
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3998..4586)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4760..5617)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVDAS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5618..5722)
                     /label=AmpR promoter
ORIGIN
        1 ttgtcatgtt tgacagctta tcttcgatta gctttaatgc ggtagtttat cacagttaaa
       61 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg
      121 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt
      181 gcgggatatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
      241 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
      301 cgggctttgt tagcagccgg atctaagctt gagctcgagt cgacccatgg aacaagaccc
      361 gggaagagaa ttcggatcct cttcccttgt cgtcgtcatc cttgtagtca gatccacgcg
      421 gaaccagaag tgccccggag gatgagattt tcttaaagcg gttggctgct gagacggcta
      481 tgaaattctt tttccatcgt cgcttcatat gtatatctcc ttcttaaagt taaacaaaat
      541 tatttctaga ggggaattgt tatccgctca caattcccct atagtgagtc gtattaattt
      601 cgcgggatcg agatctcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc
      661 acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc
      721 cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg
      781 ggactgttgg gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc
      841 ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagat
      901 cccggacacc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
      961 cagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc
     1021 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     1081 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     1141 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct
     1201 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     1261 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     1321 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     1381 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     1441 gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca
     1501 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     1561 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     1621 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     1681 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     1741 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata
     1801 cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca aacaggattt
     1861 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     1921 gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa
     1981 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     2041 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt
     2101 aggcaccggg atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt
     2161 gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg
     2221 taggacaggt gccggcagcg ctctgggtca ttttcggcga ggaccgcttt cgctggagcg
     2281 cgacgatgat cggcctgtcg cttgcggtat tcggaatctt gcacgccctc gctcaagcct
     2341 tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca ggccattatc gccggcatgg
     2401 cggccgacgc gctgggctac gtcttgctgg cgttcgcgac gcgaggctgg atggccttcc
     2461 ccattatgat tcttctcgct tccggcggca tcgggatgcc cgcgttgcag gccatgctgt
     2521 ccaggcaggt agatgacgac catcagggac agcttcaagg atcgctcgcg gctcttacca
     2581 gcctaacttc gatcactgga ccgctgatcg tcacggcgat ttatgccgcc tcggcgagca
     2641 catggaacgg gttggcatgg attgtaggcg ccgccctata ccttgtctgc ctccccgcgt
     2701 tgcgtcgcgg tgcatggagc cgggccacct cgacctgaat ggaagccggc ggcacctcgc
     2761 taacggattc accactccaa gaattggagc caatcaattc ttgcggagaa ctgtgaatgc
     2821 gcaaaccaac ccttggcaga acatatccat cgcgtccgcc atctccagca gccgcacgcg
     2881 gcgcatctcg ggcagcgttg ggtcctggcc acgggtgcgc atgatcgtgc tcctgtcgtt
     2941 gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg
     3001 cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat
     3061 ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat
     3121 tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt
     3181 attaacgaag cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac
     3241 cgccagttgt ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg
     3301 tatcgtgagc atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc
     3361 ttacacggag gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat
     3421 cagaagccag acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc
     3481 agacatctgt gaatcgcttc acgaccacgc tgatgagctt taccgcagct gcctcgcgcg
     3541 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     3601 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     3661 gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac
     3721 tatgcggcat cagagcagat tgtactgaga gtgcaccata tatgcggtgt gaaataccgc
     3781 acagatgcgt aaggagaaaa taccgcatca ggcggtcttc cgcttcctcg ctcactgact
     3841 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     3901 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     3961 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4021 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4081 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4141 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4201 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4261 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4321 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4381 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     4441 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4501 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4561 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4621 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     4681 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     4741 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     4801 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     4861 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     4921 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4981 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     5041 ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt
     5101 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     5161 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5221 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5281 ccgtaagatg cttttctgtg actggtgacg cgtcaaccaa gtcattctga gaatagtgta
     5341 tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg ccacatagca
     5401 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     5461 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     5521 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     5581 agggaataag ggcgacacgg aaatgttgaa tactcatact gttccttttt caatattatt
     5641 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     5701 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     5761 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc
     5821 aagaa
//
