LOCUS       sequence-8110-ex        5494 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5494)
  AUTHORS   Fehr M, Frommer WB, Lalonde S
  TITLE     Visualization of maltose uptake in living yeast cells by fluorescent
            nanosensors.
  JOURNAL   Proc Natl Acad Sci U S A. 2002 Jul 23. 99(15):9846-51.
  PUBMED    12097642
REFERENCE   2  (bases 1 to 5494)
  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: "Proc Natl 
            Acad Sci U S A. 2002 Jul 23. 99(15):9846-51."
COMMENT     Sequence Label: pRSET FLIPmal
FEATURES             Location/Qualifiers
     source          1..5494
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     19..38
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        19..37
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             67..89
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             109..126
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             130..162
                     /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(166..174,175..189)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(202..204,205..207,208..918)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             937..2034
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1891..1900
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2008..2031
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             join(2065..2067,2068..2070,2071..2784)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2840..2858)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2854..2901
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2998..3453
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3085..3104)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3295..3316
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3534..3638
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3639..3707,3708..4499)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCDTLLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDESDTTMPVAMPTTLRKLLTGELLTLASRQQLID*MEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKRSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      4670..5258
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5159..5178
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5412..5429
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 atctcgatcc cgcgaaatta atacgactca ctatagggag accacaacgg tttccctcta
       61 gataattttg tttaacttta agaaggagat atacatatgc ggggttctca tcatcatcat
      121 catcatggta tggctagcat gactggtgga cagcaaatgg gtcgggatct gtacgacgat
      181 gacgataagg atccgggccg catggtgagc aagggcgagg agctgttcac cggggtggtg
      241 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
      301 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
      361 ctgcccgtgc cctggcccac cctcgtgacc accctgacct ggggcgtgca gtgcttcagc
      421 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
      481 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
      541 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
      601 gacggcaaca tcctggggca caagctggag tacaactaca tcagccacaa cgtctatatc
      661 accgccgaca agcagaagaa cggcatcaag gccaacttca agatccgcca caacatcgag
      721 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
      781 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
      841 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
      901 atggacgagc tgtacaaggg tggtaccgga ggcgccaaaa tcgaagaagg taaactggta
      961 atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa gaaattcgag
     1021 aaagataccg gaattaaagt caccgttgag catccggata aactggaaga gaaattccca
     1081 caggttgcgg caactggcga tggccctgac attatcttct gggcacacga ccgctttggt
     1141 ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt ccaggacaag
     1201 ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc ttacccgatc
     1261 gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc gccaaaaacc
     1321 tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg
     1381 ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg gggttatgcg
     1441 ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg
     1501 aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa tgcagacacc
     1561 gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac catcaacggc
     1621 ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac ggtactgccg
     1681 accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc
     1741 gccagtccga acaaagagct ggcgaaagag ttcctcgaaa actatctgct gactgatgaa
     1801 ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag
     1861 gaagagttgg cgaaagatcc acgtattgcc gccaccatgg aaaacgccca gaaaggtgaa
     1921 atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc
     1981 aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca gactcgtatc
     2041 accaagggcg ccggtaccgg tggaatggtg agcaagggcg aggagctgtt caccggggtg
     2101 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     2161 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     2221 aagctgcccg tgccctggcc caccctcgtg accaccttcg gctacggcct gcagtgcttc
     2281 gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     2341 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     2401 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     2461 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     2521 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     2581 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     2641 cccgtgctgc tgcccgacaa ccactacctg agctaccagt ccgccctgag caaagacccc
     2701 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     2761 ggcatggacg agctgtacaa gtaaaagctt gatccggctg ctaacaaagc ccgaaaggaa
     2821 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     2881 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggatctgg cgtaatagcg
     2941 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg
     3001 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     3061 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3121 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagag
     3181 ctttacggca cctcgaccgc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     3241 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     3301 tcttgttcca aactggaaca acactcaacc ctatcgcggt ctattctttt gatttataag
     3361 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa atatttaacg
     3421 cgaattttaa caaaatatta acgtttacaa tttcgcctga tgcggtattt tctccttacg
     3481 catctgtgcg gtatttcaca ccgcatacag gtggcacttt tcggggaaat gtgcgcggaa
     3541 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3601 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     3661 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     3721 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     3781 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     3841 gcacttttaa agttctgcta tgtgatacac tattatcccg tattgacgcc gggcaagagc
     3901 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     3961 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     4021 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     4081 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     4141 atgaagccat accaaacgac gagagtgaca ccacgatgcc tgtagcaatg ccaacaacgt
     4201 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4261 gaatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     4321 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     4381 ggccagatgg taagcgctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     4441 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4501 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4561 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4621 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4681 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4741 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4801 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4861 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     4921 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4981 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     5041 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     5101 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     5161 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5221 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5281 tacggttcct gggcttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     5341 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     5401 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     5461 ctctccccgc gcgttggccg attcattaat gcag
//
