LOCUS       sequence-394756-        7626 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV overexpression of cyto-iAspSnFR in mammalian cell lines.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7626)
  AUTHORS   Hellweg L, Pfeifer M, Tarnawski M, Thing-Teoh S, Chang L, Bergner A,
            Kress J, Hiblot J, Wiedmer T, Superti-Furga G, Reinhardt J, Johnsson
            K, Leippe P
  TITLE     AspSnFR: A genetically encoded biosensor for real-time monitoring of
            aspartate in live cells.
  JOURNAL   Cell Chem Biol. 2024 May 20:S2451-9456(24)00179-X. doi: 
            10.1016/j.chembiol.2024.05.002.
  PUBMED    38806058
REFERENCE   2  (bases 1 to 7626)
  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: "Cell Chem 
            Biol. 2024 May 20:S2451-9456(24)00179-X. doi: 
            10.1016/j.chembiol.2024.05.002."
COMMENT     Sequence Label: pcDNA3.1/cyto-iAspSnFR
FEATURES             Location/Qualifiers
     source          1..7626
                     /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             911..1606
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(1044..1062)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1312..1331
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2572..2593
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2696..2717)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(3174..3198)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3217..3234)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3223..3447
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3493..3921
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3580..3599)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3790..3811
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3930..3950)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3935..4264
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4115..4250
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4177..4196
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4331..5125
                     /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(4385..4404)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4995..5014
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5299..5420
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5336..5355)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5390..5409
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5469..5485)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5469..5485)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5482..5504)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5493..5509
                     /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(5517..5523,5524..5541,5542..5547))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5562..5583
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5700..5717)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5871..6459)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5951..5970)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6630..7421,7422..7490))
                     /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     7253..7272
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7491..7595)
                     /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 gctggctagt
      901 taagcttacc atggtgagca agggcgaggc agtgatcaag gagttcatgc ggttcaaggt
      961 gcacatggag ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg
     1021 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
     1081 ctcctgggac atcctgtccc ctcagttcat gtacggctcc agggccttca tcaagcaccc
     1141 cgccgacatc cccgactact ataagcagtc cttccccgag ggcttcaagt gggagcgcgt
     1201 gatgaacttc gaggacggcg gcgccgtgac cgtgacccag gacacctccc tggaggacgg
     1261 caccctgatc tacaaggtga agctccgcgg caccaacttc cctcctgacg gccccgtaat
     1321 gcagaagaag acaatgggct gggaagcgtc caccgagcgg ttgtaccccg aggacggcgt
     1381 gctgaagggc gacattaaga tggccctgcg cctgaaggac ggcggccgct acctggcgga
     1441 cttcaagacc acctacaagg ccaagaagcc cgtgcagatg cccggcgcct acaacgtcga
     1501 ccgcaagttg gacatcacct cccacaacga ggactacacc gtggtggaac agtacgaacg
     1561 ctccgagggc cgccactcca ccggcggcat ggacgagctg tacaagggat ccgccgcagg
     1621 cagcacgctg gacaaaatcg ccaaaaacgg tgtgattgtc gtcggtcacc gtgaatcttc
     1681 agtgcctttc tcttattacg acaatcagca aaaagtggtg ggtttctcgc aggattactc
     1741 caacgccatt gttgaagcag tgaaaaagaa actcaacaaa ccggacttgc aggtaaaact
     1801 gattccgatt accgcgcaaa accgtattcc actgctgcaa aacggcactt tcgattttga
     1861 atgtggttct accgacaaca acgtcgaacg ccaaaaacag gcggctttct ctgacactat
     1921 tttcgtggtc accacgcgcc tgttgaccaa aaagggtggc gatatcaaag attttgccaa
     1981 cctgaaagac aaagccgtag tcgtcacttc cggcactacc tctgaagttt tgctcaacaa
     2041 actgaatgaa gagcaaaaaa tgaatatgcg catcatcagc gccaaagatc acggtgactc
     2101 tttccgcacc ctggaaagcg gtcgtgccgt tgcctttatg atggatgacg tgctgctggc
     2161 cggtgaacgt gcgcgagcga agaaaccaga caactgggaa atcgtcggca agccgcagtc
     2221 tcaggaggcc tggggttgta tgttgcgtaa agatgatccg cagttcaaaa agctgatgga
     2281 tgacaccatc gctcaggtga gaacctccgg tgaagcggaa aaatggtttg ataagtggtt
     2341 caaaaatcca attctggtga gccacaacgt ctatatcacc gccgacaagc agaagaacgg
     2401 catcaaggcg aacttcaaga tccgccacaa cgtggaggac ggcagcgtgc agctcgccga
     2461 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     2521 cctgagcacc cagtccgtgc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     2581 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggcgg
     2641 taccggaggg agcatgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     2701 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg cgcggcgagg gcgagggcga
     2761 tgccaccaac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     2821 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     2881 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     2941 caccatcagc ttcaaggacg acggcaccta caagacccgc gccgaggtga agttcgaggg
     3001 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     3061 cctggggcac aagctggagt acaactttaa caaccccctg aacatgaatt tcgaactgtc
     3121 agacgaaatg aaagcactgt tcaaagaacc gaatgacaag gcactgaaat aagacccagc
     3181 tttcttgtac aaagtggtga taaacccgct gatcagcctc gactgtgcct tctagttgcc
     3241 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     3301 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     3361 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     3421 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     3481 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3541 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3601 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3661 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     3721 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3781 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     3841 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     3901 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
     3961 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     4021 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     4081 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4141 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     4201 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     4261 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg
     4321 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     4381 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     4441 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     4501 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     4561 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     4621 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     4681 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     4741 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     4801 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     4861 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     4921 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     4981 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     5041 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     5101 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     5161 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     5221 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     5281 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     5341 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     5401 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     5461 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     5521 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     5581 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5641 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     5701 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5761 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5821 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5881 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gttagaggtg gcgaaacccg
     5941 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     6001 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     6061 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     6121 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6181 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6241 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     6301 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6361 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6421 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6481 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6541 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6601 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6661 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6721 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6781 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6841 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6901 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6961 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     7021 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     7081 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     7141 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7201 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7261 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7321 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     7381 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7441 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7501 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7561 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     7621 gacgtc
//
