LOCUS       sequence-339480-        4560 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Crowding FRET sensor with mCerulean3 and mCitrine for E. coli 
            expression. Linker comprises 2 helices and 3 GSG domains..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4560)
  AUTHORS   Boersma AJ, Zuhorn IS, Poolman B
  TITLE     A sensor for quantification of macromolecular crowding in living 
            cells.
  JOURNAL   Nat Methods. 2015 Mar;12(3):227-9, 1 p following 229. doi: 
            10.1038/nmeth.3257. Epub 2015 Feb 2.
  PUBMED    25643150
REFERENCE   2  (bases 1 to 4560)
  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: 
            "10.1038/nmeth.3257"; journalName: "Nat Methods"; date: "2015-03"; 
            volume: "12"; issue: "3"; pages: "227-9, 1 p following 229"
COMMENT     Sequence Label: pRSETA_crGE
FEATURES             Location/Qualifiers
     source          1..4560
                     /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             1497..1508
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     primer_bind     complement(1961..1979)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1975..2022
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2119..2574
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2206..2225)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2416..2437
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2600..2704
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2705..2773,2774..3565)
                     /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(2923..2942)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3736..4324
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4225..4244
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4478..4495
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct
       61 agaaataatt ttgtttaact ttaagaagga gatatacata tgcatcatca tcaccatcat
      121 gtgagcaaag gtgaagagct ctttaccggt 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 ggcagtggcg gtagtggcgc tgaggcagcc gctaaagaag ctgcggcaaa agaagcagcg
     1081 gcaaaagagg ctgcagctaa agaagccgca gcaaaagagg cagcagcaaa agcaggtagc
     1141 ggtggttcag gcggtagcgg tggctcaggt ggcagtggcg gttccatggt gtcaaaaggt
     1201 gaggaactgt ttacaggcgt ggtgccgatc ctggtagagc tggacgggga tgtgaatggc
     1261 cataaattca gcgtttcagg tgaaggtgag ggcgacgcca cgtacggaaa actgacactg
     1321 aaattcattt gcacaacagg taaactgcct gtgccttggc ctacactggt gaccaccttt
     1381 ggttatggtc tgatgtgctt tgctcgctat cctgaccaca tgaaacaaca tgatttcttt
     1441 aaatctgcca tgcctgaagg ctacgtgcaa gagcgcacca tttttttcaa agacgatggg
     1501 aattacaaaa cacgtgcgga ggtgaaattc gagggcgata cactggttaa ccgtatcgag
     1561 ctgaaaggta tcgacttcaa agaggacgga aacattctgg gtcataaact ggaatacaac
     1621 tacaacagcc ataacgtgta catcatggcc gacaaacaaa aaaacgggat taaagtgaac
     1681 ttcaaaatcc gccacaacat cgaagatggc agcgtgcagc tggccgacca ctatcaacaa
     1741 aacacaccga tcggcgacgg tcctgtactg ctgcctgaca accactatct gtcatatcag
     1801 agcgcactgt caaaagatcc taacgagaaa cgcgaccaca tggttctgct ggaattcgtg
     1861 acagccgctg gcattacact gggcatggac gagctgtaca aataaaagct tgatccggct
     1921 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     1981 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     2041 tccggatctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     2101 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2161 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2221 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2281 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2341 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2401 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2461 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     2521 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     2581 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2641 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     2701 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     2761 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     2821 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     2881 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     2941 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     3001 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     3061 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     3121 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     3181 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     3241 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     3301 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     3361 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     3421 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     3481 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     3541 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     3601 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     3661 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     3721 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     3781 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     3841 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     3901 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     3961 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     4021 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     4081 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     4141 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     4201 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     4261 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     4321 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     4381 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     4441 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     4501 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
//
