LOCUS       sequence-339481-        7150 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Crowding FRET sensor with mCerulean3 and mCitrine for mammalian cell
            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 7150)
  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 7150)
  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: pcDNA3.1_crGE
FEATURES             Location/Qualifiers
     source          1..7150
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             907..924
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1228..1247)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2301..2312
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     primer_bind     complement(2741..2758)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2747..2971
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3017..3445
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3104..3123)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3314..3335
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3454..3474)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3459..3788
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3639..3774
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3701..3720
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3855..4649
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3909..3928)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4519..4538
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4823..4944
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4860..4879)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4914..4933
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4993..5009)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4993..5009)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5006..5028)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5017..5033
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5041..5047,5048..5065,5066..5071))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5086..5107
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5224..5241)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5395..5983)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5475..5494)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6154..6945,6946..7014))
                     /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     6777..6796
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7015..7119)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 atgggccacc accaccatca ccacgtgtcc aagggcgagg aactgttcac cggcgtggtg
      961 cccatcctgg tggaactgga tggcgacgtg aacggccaca agttcagcgt gtccggcgag
     1021 ggcgaaggcg acgccacata tggcaagctg accctgaagt tcatctgcac caccggcaag
     1081 ctgcccgtgc cttggcctac cctcgtgacc acactgtctt ggggcgtgca gtgcttcgcc
     1141 agataccccg accatatgaa gcagcacgac ttcttcaaga gcgccatgcc cgagggctac
     1201 gtgcaggaac ggaccatctt ctttaaggac gacggcaact acaagaccag ggccgaagtg
     1261 aagttcgagg gcgacaccct cgtgaaccgg atcgagctga agggcatcga cttcaaagag
     1321 gacggcaaca tcctgggcca caagctggag tacaacgcca tccacggcaa cgtgtacatc
     1381 accgccgaca agcagaagaa cggcatcaag gccaacttcg gcctgaactg caacatcgag
     1441 gacggctccg tgcagctggc cgaccactac cagcagaaca cccccatcgg agatggcccc
     1501 gtgctgctgc ccgacaacca ctacctgagc acccagagca agctgagcaa ggaccccaac
     1561 gagaagcggg accacatggt gctgctcgag ttcgtgacag ccgccggaat caccctgggc
     1621 atggacgagc tgtacaaggg ctctggcggc agcggaggaa gtggcggatc aggcggcagt
     1681 gggggatctg gcgctgaagc cgctgccaaa gaagccgccg ctaaagaggc agctgcaaaa
     1741 gaggctgcag ctaaagaagc cgcagcaaaa gaagctgctg ccaaggccgg aagcggcgga
     1801 tctgggggat cagggggaag cggaggctca ggcggctcag gggctgaagc tgctgctaaa
     1861 gaggccgctg ccaaagaagc agctgccaaa gaagctgcag caaaagaggc tgccgcaaaa
     1921 gaggcagccg ccaaggctgg atctggcggc tctgggggca gtggcggctc cggcggaagc
     1981 ggaggatccg gcgtgtcaaa gggggaagaa ctgtttacag gggtggtgcc tattctggtg
     2041 gaactggacg gggatgtgaa tgggcataag ttctccgtgt ctggggaggg ggaaggggat
     2101 gctacctacg gaaaactgac actgaagttt atctgtacaa cagggaaact gcctgtgccc
     2161 tggcccacac tcgtgacaac ctttggctac ggcctgatgt gtttcgcccg ctaccctgat
     2221 catatgaagc agcatgattt tttcaaatcc gctatgcctg agggatatgt gcaggaaaga
     2281 acaattttct tcaaggatga tgggaattac aaaacacgcg ctgaagtgaa atttgagggg
     2341 gatacactcg tgaatcgcat tgaactgaag gggattgatt tcaaagagga cgggaatatt
     2401 ctggggcaca aactggagta taactacaac agccacaatg tgtatatcat ggctgataag
     2461 cagaaaaatg ggatcaaagt gaacttcaag atccggcaca atatcgagga tggaagtgtg
     2521 cagctggctg atcattatca gcagaatact cctatcggcg acggacctgt gctgctgcct
     2581 gataatcact atctgtccta ccagagcgcc ctgtccaagg accctaatga gaaaagggat
     2641 catatggtgc tgctggaatt tgtgaccgct gctggcatca cactgggaat ggatgaactg
     2701 tataagtaat ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt
     2761 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     2821 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     2881 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     2941 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     3001 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     3061 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     3121 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     3181 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     3241 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     3301 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     3361 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     3421 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     3481 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3541 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     3601 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     3661 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     3721 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     3781 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat
     3841 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     3901 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     3961 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     4021 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     4081 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     4141 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     4201 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     4261 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     4321 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     4381 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     4441 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     4501 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     4561 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     4621 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     4681 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     4741 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     4801 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     4861 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     4921 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
     4981 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     5041 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     5101 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     5161 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     5221 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     5281 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     5341 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5401 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5461 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5521 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5581 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5641 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5701 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5761 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5821 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5881 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     5941 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     6001 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     6061 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     6121 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     6181 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     6241 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     6301 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     6361 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     6421 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     6481 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     6541 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     6601 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     6661 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     6721 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     6781 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     6841 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     6901 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     6961 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     7021 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     7081 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     7141 acctgacgtc
//
