LOCUS       sequence-180854-        3632 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of a fluorescent protein, Blue Fluorescent 
            Protein (BFP).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3632)
  AUTHORS   Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien RY
  TITLE     Understanding, improving and using green fluorescent proteins.
  JOURNAL   Trends Biochem Sci. 1995 Nov;20(11):448-55.
  PUBMED    8578587
REFERENCE   2  (bases 1 to 3632)
  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"; journalName: "Trends 
            Biochem Sci."; date: "1995-11"; volume: "20(11)"; pages: "448-55"
COMMENT     Sequence Label: pNCS BFP
FEATURES             Location/Qualifiers
     source          1..3632
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     85..102
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     187..206
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        187..205
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             289..311
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             331..348
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             352..384
                     /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             join(388..396,397..411)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(418..420,421..423,424..1137)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(463..484)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1071..1092
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1200..1218)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1214..1261
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1358..1813
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1445..1464)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1655..1676
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1839..1943
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1944..2012,2013..2804)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2162..2181)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2975..3563
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3464..3483
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
       61 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
      121 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcaggat ctcgatcccg
      181 cgaaattaat acgactcact atagggagac cacaacggtt tccctctaga atcaccgagt
      241 ttattcttga cacctgatgc gatgaatgat ataataggaa agtactgttt tgtttaactt
      301 taagaaggag atatacatat gcggggttct catcatcatc atcatcatgg tatggctagc
      361 atgactggtg gacagcaaat gggtcgggat ctgtacgacg atgacgataa ggatccgatg
      421 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
      481 gacgtaaacg gccacaagtt cagcgtgagg ggcgagggcg agggcgatgc caccaacggc
      541 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
      601 gtgaccaccc tgagccacgg cgtgcagtgc ttcgcccgct accccgacca catgaagcag
      661 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
      721 aaggacgacg gcacctacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
      781 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
      841 ctggagtaca acttcaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
      901 atcaaggtga acttcaagat ccgccacaac gtggaggacg gcagcgtgca gctcgccgac
      961 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga cagccactac
     1021 ctgagcaccc agtccgtgct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1081 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaagaa
     1141 ttcgaagctt gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     1201 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     1261 gctgaaagga ggaactatat ccggatctgg cgtaatagcg aagaggcccg caccgatcgc
     1321 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta
     1381 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     1441 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     1501 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     1561 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     1621 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     1681 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     1741 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     1801 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     1861 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     1921 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     1981 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     2041 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     2101 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     2161 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     2221 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     2281 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     2341 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     2401 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     2461 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     2521 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     2581 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     2641 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     2701 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     2761 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     2821 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     2881 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     2941 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3001 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3061 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3121 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3181 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3241 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3301 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3361 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     3421 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     3481 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     3541 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     3601 tttgctggcc ttttgctcac atgttctttc ct
//
