LOCUS       sequence-314149-        6118 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ddRFP A-NES.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6118)
  AUTHORS   Ding Y, Li J, Enterina JR, Shen Y, Zhang I, Tewson PH, Mo GC, Zhang 
            J, Quinn AM, Hughes TE, Maysinger D, Alford SC, Zhang Y, Campbell RE
  TITLE     Ratiometric biosensors based on dimerization-dependent fluorescent 
            protein exchange.
  JOURNAL   Nat Methods. 2015 Jan 26. doi: 10.1038/nmeth.3261.
  PUBMED    25622108
REFERENCE   2  (bases 1 to 6118)
  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: "Nat 
            Methods. 2015 Jan 26. doi: 10.1038/nmeth.3261."
COMMENT     Sequence Label: RA-NES
FEATURES             Location/Qualifiers
     source          1..6118
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      672..681
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(880..898)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1148..1167
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1475..1492)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1481..1705
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1751..2179
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1838..1857)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2048..2069
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2188..2208)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2193..2522
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2373..2508
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2435..2454
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2589..3383
                     /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(2643..2662)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3253..3272
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3557..3678
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3594..3613)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3648..3667
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3727..3743)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3727..3743)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3740..3762)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3751..3767
                     /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(3775..3781,3782..3799,3800..3805))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3820..3841
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3958..3975)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4129..4717)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4209..4228)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4888..5679,5680..5748))
                     /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     5511..5530
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5749..5853)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5928..5947)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgtgc cgccaccatg gcctcgagaa tgaacctggt ggacctccaa aagaagctgg
      721 aggagctgga gctggacgag cagcagatgg tgagcaagag cgaggaggtc atcaaagagt
      781 tcatgcgctt caaggtgcgc ttggagggct ccatgaacgg ccacgagttc gagatcgagg
      841 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
      901 gcggccccct gcccttcgcc tgggacatcc tgtcccccca gatcatgtac ggctccaagg
      961 cgtacgtgaa gcaccccgcc gacgtccccg attacatgaa gctgtccttc cccgagggct
     1021 tcaagtggga gcgcgtgatg cacttcgagg acggcggtct ggtgaccgtg actcaggaca
     1081 cctccctgca ggacggcacg ctgatctaca aggtgaagat gcgcggcacc aacttccccc
     1141 ccgacggccc cgtaatgcag aagaagacct tgggctggga ttatgccacc gagcgcctgt
     1201 accccgaaga cggcgtgctg aagggcgagc ttctggggcg cctgaagctg aaggacggcg
     1261 gcctcaacct ggtggagttc aagaccatct acatggccaa gaagcccgtg caactgcccg
     1321 gctactactt cgtggacacc aagctgggca tcacctccca caacgaggac tacaccatcg
     1381 tggaacagta cgagcgctcc gagggccgcc accacctggg catggacgag ctgtacaagg
     1441 gtacctaata gaagcttggt aaaccgctga tcagcctcga ctgtgccttc tagttgccag
     1501 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     1561 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     1621 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     1681 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     1741 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     1801 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     1861 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     1921 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     1981 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     2041 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     2101 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     2161 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     2221 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     2281 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     2341 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     2401 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     2461 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     2521 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
     2581 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     2641 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     2701 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     2761 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     2821 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     2881 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     2941 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     3001 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     3061 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     3121 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     3181 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     3241 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     3301 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     3361 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     3421 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     3481 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     3541 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     3601 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     3661 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     3721 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     3781 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     3841 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3901 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     3961 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4021 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4081 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4141 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4201 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4261 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4321 tcatagctca cgctataggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4381 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4441 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4501 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4561 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4621 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4681 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4741 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4801 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4861 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4921 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4981 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5041 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5101 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5161 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5221 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5281 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5341 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5401 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5461 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5521 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5581 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5641 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5701 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5761 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5821 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     5881 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
     5941 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
     6001 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
     6061 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgtt
//
