LOCUS       sequence-456967-        7431 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Fluorescent reporter for malonyl-CoA that does not bind malonyl-CoA 
            (control).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7431)
  AUTHORS   Ranzau BL, Robinson TD, Scully JM, Kapelczack ED, Dean TS, TeSlaa T,
            Schmitt DL
  TITLE     A Genetically Encoded Fluorescent Biosensor for Intracellular 
            Measurement of Malonyl-CoA.
  JOURNAL   bioRxiv [Preprint]. 2024 Sep 28:2024.09.27.615526. doi: 
            10.1101/2024.09.27.615526.
  PUBMED    39386450
REFERENCE   2  (bases 1 to 7431)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Sep 28:2024.09.27.615526. doi: 
            10.1101/2024.09.27.615526."
COMMENT     Sequence Label: pcDNA-deadMalibu-mScarlet
FEATURES             Location/Qualifiers
     source          1..7431
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1211..1230)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1402..1781
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1782..1985
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1936..1956
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2030..2049
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2030..2048
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2068..2077
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2092..2124
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(2128..2136,2137..2151)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     2442..2463
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2569..2590)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2830..2849)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     regulatory      3385..3394
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3391..4086
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(3524..3542)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3792..3811
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(4150..4168)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4151..4168)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4188..4205)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4194..4418
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4464..4892
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4551..4570)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4761..4782
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4901..4921)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4906..5235
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5086..5221
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5148..5167
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5302..6096
                     /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(5356..5375)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5966..5985
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6270..6392
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6307..6326)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6362..6381
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6441..6457)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6441..6457)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6454..6476)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6465..6481
                     /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(6489..6495,6496..6513,6514..6519))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6534..6555
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6672..6689)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6843..7431)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6923..6942)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     1201 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     1261 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     1321 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     1381 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
     1441 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     1501 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     1561 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     1621 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     1681 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     1741 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     1801 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     1861 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     1921 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     1981 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
     2041 actataggga gacccaagct tgcggccgcc accatggtgc ggggttctgg tatggctagc
     2101 atgactggtg gacagcaaat gggtcgggat ctgtacgacg atgacgataa ggatcccatg
     2161 gagctttcca tacctgaact gagagaaaga attaagaacg tggcagagaa aacacttgag
     2221 gacgaatcag gtaggaacgt ctatatcaag gccgacaagc agaagaacgg catcaaggcg
     2281 aacttcaaga tccgccacaa catcgaggac ggcggcgtgc agctcgccta ccactaccag
     2341 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcgtg
     2401 cagtccaaac tttcgaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2461 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggcgg taccggaggg
     2521 agcatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     2581 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggtgaggg cgatgccacc
     2641 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     2701 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     2761 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acatccagga gcgcaccatc
     2821 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     2881 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     2941 cacaagctgg agtacaacat tgaggtgaag tccctgtcac ttgatgaagt tatcggagaa
     3001 attattgacc ttgagctgga tgatcaggcg atatccattt tagaaataaa acaggagcac
     3061 gtgttcagcc ggaatcagat tgcggcagga caccatttat ttgcacaggc gaactctttg
     3121 gccgttgcag tcattgatga cgagctggcg ctgactgcaa gtgcagacat ccgctttaca
     3181 agacaggtaa agcagggtga acgtgtcgta gcaaaagcga aagtgacggc tgtcgaaaaa
     3241 gaaaaaggaa gaacggttgt cgaagtgaac agctacgttg gcgaagaaat tgttttttct
     3301 ggacgctttg acatgtatcg ttcaaaacat tcactgcaga gcaccggcag cggcaacgcg
     3361 gtgggccagg atacccagga acgcgccacc atggtgagca agggcgaggc agtgatcaag
     3421 gagttcatgc ggttcaaggt gcacatggag ggctccatga acggccacga gttcgagatc
     3481 gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc
     3541 aagggtggcc ccctgccctt ctcctgggac atcctgtccc ctcagttcat gtacggctcc
     3601 agggccttca ccaagcaccc cgccgacatc cccgactact ataagcagtc cttccccgag
     3661 ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgccgtgac cgtgacccag
     3721 gacacctccc tggaggacgg caccctgatc tacaaggtga agctccgcgg caccaacttc
     3781 cctcctgacg gccccgtaat gcagaagaag acaatgggct gggaagcgtc caccgagcgg
     3841 ttgtaccccg aggacggcgt gctgaagggc gacattaaga tggccctgcg cctgaaggac
     3901 ggcggccgct acctggcgga cttcaagacc acctacaagg ccaagaagcc cgtgcagatg
     3961 cccggcgcct acaacgtcga ccgcaagttg gacatcacct cccacaacga ggactacacc
     4021 gtggtggaac agtacgaacg ctccgagggc cgccactcca ccggcggcat ggacgagctg
     4081 tacaagtaag aattctgcag atatccatca cactggcggc cgctcgagca tgcatctaga
     4141 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc
     4201 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     4261 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     4321 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     4381 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     4441 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4501 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     4561 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4621 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     4681 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4741 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     4801 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     4861 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     4921 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     4981 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     5041 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     5101 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     5161 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     5221 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     5281 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     5341 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     5401 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     5461 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     5521 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     5581 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     5641 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     5701 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     5761 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     5821 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     5881 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     5941 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     6001 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     6061 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     6121 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     6181 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     6241 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     6301 aataaagcaa tagcatcaca aatttcacaa ataaagcatt ttttttcact gcattctagt
     6361 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     6421 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     6481 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     6541 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     6601 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     6661 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     6721 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     6781 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     6841 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     6901 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     6961 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     7021 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     7081 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     7141 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     7201 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     7261 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     7321 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     7381 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca a
//
