LOCUS       sequence-8111-ex        5479 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 5479)
  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 5479)
  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-2u
FEATURES             Location/Qualifiers
     source          1..5479
                     /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"
     regulatory      1876..1885
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1993..2016
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             join(2050..2052,2053..2055,2056..2769)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2825..2843)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2839..2886
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2983..3438
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3070..3089)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3280..3301
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3519..3623
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3624..3692,3693..4484)
                     /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      4655..5243
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5144..5163
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5397..5414
                     /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 ggcgccaaac tggtaatctg gattaacggc
      961 gataaaggct ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt
     1021 aaagtcaccg ttgagcatcc ggataaactg gaagagaaat tcccacaggt tgcggcaact
     1081 ggcgatggcc ctgacattat cttctgggca cacgaccgct ttggtggcta cgctcaatct
     1141 ggcctgttgg ctgaaatcac cccggacaaa gcgttccagg acaagctgta tccgtttacc
     1201 tgggatgccg tacgttacaa cggcaagctg attgcttacc cgatcgctgt tgaagcgtta
     1261 tcgctgattt ataacaaaga tctgctgccg aacccgccaa aaacctggga agagatcccg
     1321 gcgctggata aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa
     1381 ccgtacttca cctggccgct gattgctgct gacgggggtt atgcgttcaa gtatgaaaac
     1441 ggcaagtacg acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc
     1501 ttcctggttg acctgattaa aaacaaacac atgaatgcag acaccgatta ctccatcgca
     1561 gaagctgcct ttaataaagg cgaaacagcg atgaccatca acggcccgtg ggcatggtcc
     1621 aacatcgaca ccagcaaagt gaattatggt gtaacggtac tgccgacctt caagggtcaa
     1681 ccatccaaac cgttcgttgg cgtgctgagc gcaggtatta acgccgccag tccgaacaaa
     1741 gagctggcga aagagttcct cgaaaactat ctgctgactg atgaaggtct ggaagcggtt
     1801 aataaagaca aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa
     1861 gatccacgta ttgccgccac catggaaaac gcccagaaag gtgaaatcat gccgaacatc
     1921 ccgcagatgt ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt
     1981 cgtcagactg tcgatgaagc cctgaaagac gcgcagactc gtatcaccaa gggcgccggt
     2041 accggtggaa tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     2101 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     2161 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     2221 tggcccaccc tcgtgaccac cttcggctac ggcctgcagt gcttcgcccg ctaccccgac
     2281 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     2341 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     2401 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     2461 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     2521 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     2581 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     2641 gacaaccact acctgagcta ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     2701 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     2761 tacaagtaaa agcttgatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     2821 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     2881 tttttgctga aaggaggaac tatatccgga tctggcgtaa tagcgaagag gcccgcaccg
     2941 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
     3001 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     3061 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     3121 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagagcttta cggcacctcg
     3181 accgcaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     3241 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     3301 gaacaacact caaccctatc gcggtctatt cttttgattt ataagggatt ttgccgattt
     3361 cggcctattg gttaaaaaat gagctgattt aacaaatatt taacgcgaat tttaacaaaa
     3421 tattaacgtt tacaatttcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3481 tcacaccgca tacaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3541 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3601 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3661 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3721 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3781 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3841 tgctatgtga tacactatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     3901 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3961 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     4021 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     4081 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     4141 acgacgagag tgacaccacg atgcctgtag caatgccaac aacgttgcgc aaactattaa
     4201 ctggcgaact acttactcta gcttcccggc aacaattaat agactgaatg gaggcggata
     4261 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4321 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     4381 gctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4441 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4501 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4561 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4621 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4681 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4741 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4801 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4861 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4921 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4981 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5041 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5101 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5161 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5221 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctgggct
     5281 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5341 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5401 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     5461 ggccgattca ttaatgcag
//
