LOCUS       sequence-340008-        4410 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Crowding FRET sensor with mCerulean3 and mCitrine for E. coli 
            expression. Linker comprises 1 helix and 2 GSG linkers.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4410)
  AUTHORS   Liu B, Aberg C, van Eerden FJ, Marrink SJ, Poolman B, Boersma AJ
  TITLE     Design and Properties of Genetically Encoded Probes for Sensing 
            Macromolecular Crowding.
  JOURNAL   Biophys J. 2017 May 9;112(9):1929-1939. doi: 
            10.1016/j.bpj.2017.04.004.
  PUBMED    28494963
REFERENCE   2  (bases 1 to 4410)
  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: 
            "10.1016/j.bpj.2017.04"; journalName: "Biophys J"; date: "2017-05-9-
            9"; volume: "112"; issue: "9"; pages: "1929-1939"
COMMENT     Sequence Label: pRSETA_crE6
FEATURES             Location/Qualifiers
     source          1..4410
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..39
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             70..92
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             103..120
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1347..1358
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     primer_bind     complement(1811..1829)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1825..1872
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1969..2424
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2056..2075)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2266..2287
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2450..2554
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2555..2623,2624..3415)
                     /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(2773..2792)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3586..4174
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4075..4094
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4328..4345
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct
       61 agaaataatt ttgtttaact ttaagaagga gatatacata tgcatcatca tcaccatcat
      121 gtgagcaaag gtgaagaact gtttaccggt gttgttccga ttctggttga actggatggt
      181 gacgttaatg gtcacaaatt ttcagttagc ggtgaaggcg aaggtgatgc aacctatggt
      241 aaactgaccc tgaaatttat ctgtaccacc ggcaaactgc cggttccgtg gccgaccctg
      301 gttaccaccc tgagctgggg tgttcagtgt tttgcacgtt atccggatca catgaaacag
      361 cacgattttt tcaaaagcgc aatgccggaa ggttatgttc aagaacgtac catcttcttc
      421 aaagatgacg gcaactataa aacccgtgcc gaagttaaat ttgaaggtga taccctggtg
      481 aatcgcattg aactgaaagg catcgatttt aaagaggatg gtaatatcct gggccacaaa
      541 ctggaatata atgccattca tggcaacgtg tatatcaccg cagataaaca gaaaaacggc
      601 atcaaagcaa attttggcct gaactgcaat attgaagatg gttcagttca gctggcagat
      661 cattatcagc agaatacccc gattggtgat ggtccggttc tgctgccgga taatcattat
      721 ctgagcaccc agagcaaact gagcaaagat ccgaatgaaa aacgtgatca catggtgctg
      781 ctggaatttg ttaccgcagc aggtattacc ctgggtatgg atgaactgta taaaggtagc
      841 ggtggtagtg gtggttcagg tggctctggt ggcagcggtg gatccggtgc agaagcagca
      901 gcaaaagaag ccgctgccaa agaagcggca gcgaaagagg ctgccgcaaa agaggcagca
      961 gcgaaagaag cagcggctaa agcaggttca ggcggttctg ggggttctgg cggtagcgga
     1021 ggctcaggtg gttccatggt gtcaaaaggc gaggaactgt tcacaggcgt ggtgccgatc
     1081 ctggtagagc tggacgggga tgtgaatggc cataaattca gcgtttcagg tgaaggtgag
     1141 ggcgacgcca cgtacggaaa actgacactg aaattcattt gcacaacagg taaactgcct
     1201 gtgccttggc ctacactggt gaccaccttt ggttatggtc tgatgtgctt tgctcgctat
     1261 cctgaccaca tgaaacaaca tgatttcttt aaatctgcca tgcctgaagg ctacgtgcaa
     1321 gagcgcacca tttttttcaa agacgatggg aattacaaaa cacgtgcgga ggtgaaattc
     1381 gagggcgata cactggttaa ccgtatcgag ctgaaaggta tcgacttcaa agaggacgga
     1441 aacattctgg gtcataaact ggaatacaac tacaacagcc ataacgtgta catcatggcc
     1501 gacaaacaaa aaaacgggat taaagtgaac ttcaaaatcc gccacaacat cgaagatggc
     1561 agcgtgcagc tggccgacca ctatcaacaa aacacaccga tcggcgacgg tcctgtgctg
     1621 ctgccagaca accactatct gtcatatcag agcgcactgt caaaagatcc taacgagaaa
     1681 cgcgaccaca tggttctgct ggaattcgtg acagccgctg gcattacact gggcatggac
     1741 gagctgtaca aataaaagct tgatccggct gctaacaaag cccgaaagga agctgagttg
     1801 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1861 aggggttttt tgctgaaagg aggaactata tccggatctg gcgtaatagc gaagaggccc
     1921 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta
     1981 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2041 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2101 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2161 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2221 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2281 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     2341 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     2401 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     2461 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     2521 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     2581 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     2641 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     2701 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     2761 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     2821 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     2881 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     2941 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     3001 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     3061 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     3121 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     3181 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     3241 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     3301 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     3361 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     3421 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     3481 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     3541 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3601 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3661 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3721 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3781 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3841 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3901 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3961 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     4021 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     4081 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     4141 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     4201 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     4261 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     4321 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     4381 ccccgcgcgt tggccgattc attaatgcag
//
