LOCUS       sequence-457487-        5429 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of EBFP2 blue fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
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 May 22;46(20):5904-10. doi: 10.1021/bi700199g
  PUBMED    17444659
REFERENCE   2  (bases 1 to 5429)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10"; journalName: 
            "Biochemistry"; date: "2007-05-22- 22"; volume: "46"; issue: "20"; 
            pages: "5904-10"
COMMENT     Sequence Label: pBad-EBFP2
FEATURES             Location/Qualifiers
     source          1..5429
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      10..96
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      188..215
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        234..325
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(326..394,395..1186)
                     /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(544..563)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1228..1683
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1315..1334)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1525..1546
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      1794..2382
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2283..2302
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2536..2553
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2568..2708
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2634..2653)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2794..2816
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2954..2973
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(3153..4031)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPPRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        4058..4342
                     /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     4265..4284
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             4369..4391
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4411..4428
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4432..4464
                     /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             4477..4497
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(4504..4506,4507..4509,4510..5223)
                     /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(4549..4570)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5269..5286)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
ORIGIN
        1 gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
       61 tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt
      121 gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa
      181 attaagcaga aggccatcct gacggatggc ctttttgcgt ttctacaaac tcttttgttt
      241 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
      301 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
      361 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
      421 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
      481 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
      541 tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg
      601 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
      661 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
      721 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
      781 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
      841 aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt
      901 aactggcgaa ccacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
      961 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     1021 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     1081 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     1141 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     1201 ttactcatat atactttaga ttgatttacg cgccctgtag cggcgcatta agcgcggcgg
     1261 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     1321 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     1381 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     1441 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     1501 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     1561 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     1621 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     1681 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     1741 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     1801 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     1861 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     1921 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     1981 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     2041 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     2101 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     2161 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     2221 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     2281 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     2341 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     2401 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     2461 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     2521 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     2581 tttctcctta cgcatctgtg cggtatttca caccgcatct ggtgcactct cagtacaatc
     2641 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     2701 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     2761 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     2821 caccgtcatc accgaaacgc gcgaggcaga aggagatggc gcccaacagt cccccggcca
     2881 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     2941 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     3001 tgatgccggc cacgatgcgt ccggcgtaga ggatctgctc atgtttgaca gcttatcatc
     3061 gatgcataat gtgcctgtca aatggacgaa gcagggattc tgcaaaccct atgctactcc
     3121 gtcaagccgt caattgtctg attcgttacc aattatgaca acttgacggc tacatcattc
     3181 actttttctt cacaaccggc acggaactcg ctcgggctgg ccccggtgca ttttttaaat
     3241 acccgcgaga aatagagttg atcgtcaaaa ccaacattgc gaccgacggt ggcgataggc
     3301 atccgggtgg tgctcaaaag cagcttcgcc tggctgatac gttggtcctc gcgccagctt
     3361 aagacgctaa tccctaactg ctggcggaaa agatgtgaca gacgcggcgg cgacaagcaa
     3421 acatgctgtg cgacgctggc gatatcaaaa ttgctgtctg ccaggtgatc gctgatgtac
     3481 tgacaagcct cgcgtacccg attatccatc ggtggatgga gcgactcgtt aatcgcttcc
     3541 atgcgccgca gtaacaattg ctcaagcaga tttatcgcca gcagctccga atagcgccct
     3601 tccccttgcc cggcgttaat gatttgccca aacaggtcgc tgaaatgcgg ctggtgcgct
     3661 tcatccgggc gaaagaaccc cgtattggca aatattgacg gccagttaag ccattcatgc
     3721 cagtaggcgc gcggacgaaa gtaaacccac tggtgatacc attcgcgagc ctccggatga
     3781 cgaccgtagt gatgaatctc tcctggcggg aacagcaaaa tatcacccgg tcggcaaaca
     3841 aattctcgtc cctgattttt caccaccccc tgaccgcgaa tggtgagatt gagaatataa
     3901 cctttcattc ccagcggtcg gtcgataaaa aaatcgagat aaccgttggc ctcaatcggc
     3961 gttaaacccg ccaccagatg ggcattaaac gagtatcccg gcagcagggg atcattttgc
     4021 gcttcagcca tacttttcat actcccgcca ttcagagaag aaaccaattg tccatattgc
     4081 atcagacatt gccgtcactg cgtcttttac tggctcttct cgctaaccaa accggtaacc
     4141 ccgcttatta aaagcattct gtaacaaagc gggaccaaag ccatgacaaa aacgcgtaac
     4201 aaaagtgtct ataatcacgg cagaaaagtc cacattgatt atttgcacgg cgtcacactt
     4261 tgctatgcca tagcattttt atccataaga ttagcggata ctacctgacg ctttttatcg
     4321 caactctcta ctgtttctcc atacccgttt ttttgggcta gaaataattt tgtttaactt
     4381 taagaaggag atatacatat gcggggttct catcatcatc atcatcatgg tatggctagc
     4441 atgactggtg gacagcaaat gggtcgggat ctgtacgaga acctgtactt ccagggctcg
     4501 agcatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     4561 gacggcgacg taaacggcca caagttcagc gtgaggggcg agggcgaggg cgatgccacc
     4621 aacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     4681 accctcgtga ccaccctgag ccacggcgtg cagtgcttcg cccgctaccc cgaccacatg
     4741 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     4801 ttcttcaagg acgacggcac ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     4861 ctggtgaacc gcatcgagct gaagggcgtc gacttcaagg aggacggcaa catcctgggg
     4921 cacaagctgg agtacaactt caacagccac aacatctata tcatggccgt caagcagaag
     4981 aacggcatca aggtgaactt caagatccgc cacaacgtgg aggacggcag cgtgcagctc
     5041 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacagc
     5101 cactacctga gcacccagtc cgtgctgagc aaagacccca acgagaagcg cgatcacatg
     5161 gtcctgctgg agttccgcac cgccgccggg atcactctcg gcatggacga gctgtacaag
     5221 taagaattcg aagcttggct gttttggcgg atgagagaag attttcagcc tgatacagat
     5281 taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt
     5341 ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt
     5401 ggggtctccc catgcgagag tagggaact
//
