LOCUS       pBad-EBFP2                5429 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  Bacterial expression of EBFP2 blue fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    pBad-EBFP2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5429)
  AUTHORS   Ai HW, Shaner NC, Cheng Z, Tsien RY, Campbell RE
  TITLE     Exploration of New Chromophore Structures Leads to the 
            Identification of Improved Blue Fluorescent Proteins.
  JOURNAL   Biochemistry. 2007 Apr 20. ():.
  PUBMED    17444659
REFERENCE   2  (bases 1 to 5429)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            //www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5429
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             complement(53..931)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPPRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        958..1242
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1165..1184
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             1269..1291
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1311..1328
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1332..1364
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             1377..1397
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1404..2123
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(1449..1470)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2169..2186)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2339..2425
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2517..2544
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2563..2654
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2655..3515
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGEPLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2873..2892)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3557..4012
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3644..3663)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3854..3875
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      4123..4711
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4612..4631
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4865..4882
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4897..5037
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4963..4982)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5123..5145
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5283..5302
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tgcaaaccct atgctactcc gtcaagccgt caattgtctg attcgttacc aattatgaca
       61 acttgacggc tacatcattc actttttctt cacaaccggc acggaactcg ctcgggctgg
      121 ccccggtgca ttttttaaat acccgcgaga aatagagttg atcgtcaaaa ccaacattgc
      181 gaccgacggt ggcgataggc atccgggtgg tgctcaaaag cagcttcgcc tggctgatac
      241 gttggtcctc gcgccagctt aagacgctaa tccctaactg ctggcggaaa agatgtgaca
      301 gacgcggcgg cgacaagcaa acatgctgtg cgacgctggc gatatcaaaa ttgctgtctg
      361 ccaggtgatc gctgatgtac tgacaagcct cgcgtacccg attatccatc ggtggatgga
      421 gcgactcgtt aatcgcttcc atgcgccgca gtaacaattg ctcaagcaga tttatcgcca
      481 gcagctccga atagcgccct tccccttgcc cggcgttaat gatttgccca aacaggtcgc
      541 tgaaatgcgg ctggtgcgct tcatccgggc gaaagaaccc cgtattggca aatattgacg
      601 gccagttaag ccattcatgc cagtaggcgc gcggacgaaa gtaaacccac tggtgatacc
      661 attcgcgagc ctccggatga cgaccgtagt gatgaatctc tcctggcggg aacagcaaaa
      721 tatcacccgg tcggcaaaca aattctcgtc cctgattttt caccaccccc tgaccgcgaa
      781 tggtgagatt gagaatataa cctttcattc ccagcggtcg gtcgataaaa aaatcgagat
      841 aaccgttggc ctcaatcggc gttaaacccg ccaccagatg ggcattaaac gagtatcccg
      901 gcagcagggg atcattttgc gcttcagcca tacttttcat actcccgcca ttcagagaag
      961 aaaccaattg tccatattgc atcagacatt gccgtcactg cgtcttttac tggctcttct
     1021 cgctaaccaa accggtaacc ccgcttatta aaagcattct gtaacaaagc gggaccaaag
     1081 ccatgacaaa aacgcgtaac aaaagtgtct ataatcacgg cagaaaagtc cacattgatt
     1141 atttgcacgg cgtcacactt tgctatgcca tagcattttt atccataaga ttagcggata
     1201 ctacctgacg ctttttatcg caactctcta ctgtttctcc atacccgttt ttttgggcta
     1261 gaaataattt tgtttaactt taagaaggag atatacatat gcggggttct catcatcatc
     1321 atcatcatgg tatggctagc atgactggtg gacagcaaat gggtcgggat ctgtacgaga
     1381 acctgtactt ccagggctcg agcatggtga gcaagggcga ggagctgttc accggggtgg
     1441 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgaggggcg
     1501 agggcgaggg cgatgccacc aacggcaagc tgaccctgaa gttcatctgc accaccggca
     1561 agctgcccgt gccctggccc accctcgtga ccaccctgag ccacggcgtg cagtgcttcg
     1621 cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     1681 acgtccagga gcgcaccatc ttcttcaagg acgacggcac ctacaagacc cgcgccgagg
     1741 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcgtc gacttcaagg
     1801 aggacggcaa catcctgggg cacaagctgg agtacaactt caacagccac aacatctata
     1861 tcatggccgt caagcagaag aacggcatca aggtgaactt caagatccgc cacaacgtgg
     1921 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     1981 ccgtgctgct gcccgacagc cactacctga gcacccagtc cgtgctgagc aaagacccca
     2041 acgagaagcg cgatcacatg gtcctgctgg agttccgcac cgccgccggg atcactctcg
     2101 gcatggacga gctgtacaag taagaattcg aagcttggct gttttggcgg atgagagaag
     2161 attttcagcc tgatacagat taaatcagaa cgcagaagcg gtctgataaa acagaatttg
     2221 cctggcggca gtagcgcggt ggtcccacct gaccccatgc cgaactcaga agtgaaacgc
     2281 cgtagcgccg atggtagtgt ggggtctccc catgcgagag tagggaactg ccaggcatca
     2341 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
     2401 gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg
     2461 gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa
     2521 ggccatcctg acggatggcc tttttgcgtt tctacaaact cttttgttta tttttctaaa
     2581 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     2641 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     2701 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     2761 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     2821 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     2881 gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt
     2941 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     3001 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     3061 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     3121 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     3181 gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac
     3241 cacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     3301 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     3361 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     3421 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     3481 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     3541 tactttagat tgatttacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     3601 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     3661 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     3721 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     3781 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     3841 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc
     3901 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     3961 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat ttaaaaggat
     4021 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     4081 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4141 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4201 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4261 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4321 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4381 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     4441 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     4501 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     4561 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     4621 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     4681 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     4741 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     4801 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     4861 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     4921 gcatctgtgc ggtatttcac accgcatctg gtgcactctc agtacaatct gctctgatgc
     4981 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     5041 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     5101 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     5161 ccgaaacgcg cgaggcagaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     5221 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     5281 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     5341 acgatgcgtc cggcgtagag gatctgctca tgtttgacag cttatcatcg atgcataatg
     5401 tgcctgtcaa atggacgaag cagggattc
//