LOCUS       sequence-7668-ex        7717 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7717)
  AUTHORS   Okumoto S, Looger LL, Micheva KD, Reimer RJ, Smith SJ, Frommer WB
  TITLE     Detection of glutamate release from neurons by genetically encoded 
            surface-displayed FRET nanosensors.
  JOURNAL   Proc Natl Acad Sci U S A. 2005 Jun 14. 102(24):8740-5.
  PUBMED    15939876
REFERENCE   2  (bases 1 to 7717)
  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: "Proc Natl 
            Acad Sci U S A. 2005 Jun 14. 102(24):8740-5."
COMMENT     Sequence Label: pcDNA3.1 FLIPE-10u
FEATURES             Location/Qualifiers
     source          1..7717
                     /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             join(990..992,993..995,996..1706)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2565..2567,2568..2570,2571..3284)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3311..3328)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3317..3541
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3587..4015
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3674..3693)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3884..3905
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4024..4044)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4029..4358
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4209..4344
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4271..4290
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4425..5219
                     /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(4479..4498)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5089..5108
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5393..5514
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5430..5449)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5484..5503
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5563..5579)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5563..5579)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5576..5598)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5587..5603
                     /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(5611..5617,5618..5635,5636..5641))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5656..5677
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5794..5811)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5965..6550)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6045..6064)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6721..7512,7513..7581))
                     /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     7344..7363
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7582..7686)
                     /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 gtttaaacgg gccctctaga ctcgagcggc cgccactgtg ctggatatct gcagaattcc
      961 accacactgg actagtggat ccgggccgca tggtgagcaa gggcgaggag ctgttcaccg
     1021 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     1081 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     1141 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctgg ggcgtgcagt
     1201 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1261 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     1321 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1381 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacatc agccacaacg
     1441 tctatatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag atccgccaca
     1501 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1561 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     1621 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     1681 ctctcggcat ggacgagctg tacaagggtg gtaccggagg cgccgcaggc agcacgctgg
     1741 acaaaatcgc caaaaacggt gtgattgtcg tcggtcaccg tgaatcttca gtgcctttct
     1801 cttattacga caatcagcaa aaagtggtgg gttactcgca ggattactcc aacgccattg
     1861 ttgaagcagt gaaaaagaaa ctcaacaaac cggacttgca ggtaaaactg attccgatta
     1921 cctcacaaaa ccgtattcca ctgctgcaaa acggcacttt cgattttgaa tgtggttcta
     1981 ccaccaacaa cgtcgaacgc caaaaacagg cggctttctc tgacactatt ttcgtggtcg
     2041 gtacgcgcct gttgaccaaa aagggtggcg atatcaaaga ttttgccaac ctgaaagaca
     2101 aagccgtagt cgtcacttcc ggcactacct ctgaagtttt gctcaacaaa ctgaatgaag
     2161 agcaaaaaat gaatatgcgc atcatcagcg ccaaagatca cggtgactct ttccgcaccc
     2221 tggaaagcgg tcgtgccgtt gcctttatga tggatgaccg gctgctggcc ggtgaacgtg
     2281 cgaaagcgaa gaaaccagac aactgggaaa tcgtcggcaa gccgcagtct caggaggcct
     2341 acggttgtat gttgcgtaaa gatgatccgc agttcaaaaa gctgatggat gacaccatcg
     2401 ctcaggtgca gacctccggt gaagcggaaa aatggtttga taagtggttc aaaaatccaa
     2461 ttccgccgaa aaacctgaac atgaatttcg aactgtcaga cgaaatgaaa gcactgttca
     2521 aagaaccgaa tgacaaggca ctgaacggcg ccggtaccgg tggaatggtg agcaagggcg
     2581 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     2641 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     2701 agctgatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctgg
     2761 gctacggcct gcagtgcttc gcccgctacc ccgaccacat gaagcagcac gacttcttca
     2821 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     2881 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     2941 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     3001 acaacagcca caacgtctat atcaccgccg acaagcagaa gaacggcatc aaggccaact
     3061 tcaagatccg ccacaacatc gaggacggcg gcgtgcagct cgccgaccac taccagcaga
     3121 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agctaccagt
     3181 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     3241 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaaagctt aagtttaaac
     3301 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     3361 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     3421 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     3481 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     3541 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     3601 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3661 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3721 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3781 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     3841 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3901 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     3961 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     4021 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     4081 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     4141 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     4201 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     4261 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     4321 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     4381 ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     4441 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     4501 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     4561 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     4621 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     4681 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     4741 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     4801 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     4861 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4921 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     4981 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     5041 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     5101 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     5161 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     5221 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     5281 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     5341 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     5401 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     5461 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     5521 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     5581 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5641 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5701 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5761 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5821 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5881 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5941 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     6001 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     6061 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     6121 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     6181 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     6241 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     6301 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     6361 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     6421 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6481 acaaaccacc gctggtagcg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     6541 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     6601 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     6661 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6721 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     6781 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6841 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6901 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6961 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     7021 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     7081 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     7141 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     7201 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     7261 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     7321 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     7381 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     7441 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     7501 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     7561 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     7621 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     7681 tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
