LOCUS       sequence-314148-        6094 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ddGFP B-NES.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6094)
  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 6094)
  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: GB-NES
FEATURES             Location/Qualifiers
     source          1..6094
                     /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(829..847)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1097..1116
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1291..1314
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(1451..1468)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1457..1681
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1727..2155
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1814..1833)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2024..2045
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2164..2184)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2169..2498
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2349..2484
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2411..2430
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2565..3359
                     /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(2619..2638)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3229..3248
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3533..3654
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3570..3589)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3624..3643
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3703..3719)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3703..3719)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3716..3738)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3727..3743
                     /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(3751..3757,3758..3775,3776..3781))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3796..3817
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3934..3951)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4105..4693)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4185..4204)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4864..5655,5656..5724))
                     /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     5487..5506
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5725..5829)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5904..5923)
                     /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 gcctcgagag cctcctccga ggacaacaac atggccacca
      721 tcaaagagtt catgcgcttc aaggtgcgca tggagggctc catgaacggc cacgagttcg
      781 agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg
      841 tgaccaaggg cggccccctg ccctttgcct gggacatcct gtccccccag atcatgtacg
      901 gctccaaagc gtacgtgagg caccccgccg acatccccga ttacaagaag ctgcccttcc
      961 ccgagggctt caagtgggag cgcgtgatga acttcgaaga cggcggtctg gtgaccgtta
     1021 cccaggactc ctccctgcag gacggcacgc tgatctgcaa ggtgaagatt cgcggcacca
     1081 acttcccccc cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctccaccg
     1141 agatgctgta ccccaaggac ggcgtgctga agggccatag ctatcaggcc ctgaagctga
     1201 aggacggcgg ccactacctg gtggagttcg agaccatcta catggccaag aagcccgtgc
     1261 aactgcccgg cgattactgt gtggacacca agctggacat cacctcccac aacgaggact
     1321 acaccatcgt ggaacagtac gggcgctccg agggccgcca ccgtctgggc atggacgagc
     1381 ggtacaagtt agccttgaaa ttagcaggtc ttgatatcgg gagctaaaag cttggtaaac
     1441 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     1501 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     1561 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     1621 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     1681 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     1741 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     1801 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     1861 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     1921 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     1981 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     2041 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     2101 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     2161 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     2221 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     2281 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     2341 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     2401 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     2461 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     2521 ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     2581 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     2641 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     2701 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     2761 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     2821 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     2881 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     2941 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     3001 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     3061 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     3121 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     3181 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     3241 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     3301 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     3361 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     3421 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     3481 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     3541 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     3601 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     3661 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     3721 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     3781 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     3841 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3901 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     3961 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4021 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4081 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4141 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4201 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4261 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct ataggtatct
     4321 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4381 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4441 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4501 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     4561 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4621 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4681 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4741 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     4801 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     4861 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     4921 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     4981 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5041 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5101 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5161 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5221 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5281 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5341 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5401 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5461 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5521 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5581 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     5641 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     5701 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     5761 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     5821 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc
     5881 gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     5941 ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca
     6001 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
     6061 ctgcttcgcg atgtacgggc cagatatacg cgtt
//
