LOCUS       sequence-231761-        7651 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express in mammalian cells a lactate FRET sensor negative 
            control..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7651)
  AUTHORS   Baeza-Lehnert F, Flores CA, Guequen A, Barros LF
  TITLE     Monitoring Lactate Dynamics in Individual Macrophages with a 
            Genetically Encoded Probe.
  JOURNAL   Methods Mol Biol. 2020;2184:19-30. doi: 10.1007/978-1-0716-0802-9_2.
  PUBMED    32808215
REFERENCE   2  (bases 1 to 7651)
  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: "Methods Mol
            Biol."; date: "2020"; volume: "2184:19-30. doi"; pages: " 
            10.1007/978-1-0716-0802-9_2"
COMMENT     Sequence Label: Dead-Laconic/pcDNA3.1(-)
FEATURES             Location/Qualifiers
     source          1..7651
                     /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             987..1694
                     /codon_start=1
                     /product="monomeric teal (cyan) fluorescent protein (Ai et 
                     al., 2006)"
                     /label=mTFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFAYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKKTTGWDASTERMYVRDGVLK
                     GDVKHKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYESA
                     VARNSTDGMDELYK"
     CDS             join(2496..2498,2499..2501,2502..3215)
                     /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(2541..2562)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2802..2821)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3149..3170
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3242..3259)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3248..3472
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3518..3946
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3605..3624)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3815..3836
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3955..3975)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3960..4289
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4140..4275
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4202..4221
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4356..5150
                     /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(4410..4429)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5020..5039
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5324..5445
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5361..5380)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5415..5434
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5494..5510)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5494..5510)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5507..5529)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5518..5534
                     /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(5542..5548,5549..5566,5567..5572))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5587..5608
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5725..5742)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5896..6484)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5976..5995)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6655..7446,7447..7515))
                     /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     7278..7297
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7516..7620)
                     /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 ccgaacatgg tgagcaaggg cgaggagacc acaatgggcg
     1021 taatcaagcc cgacatgaag atcaagctga agatggaggg caacgtgaat ggccacgcct
     1081 tcgtgatcga gggcgagggc gagggcaagc cctacgacgg caccaacacc atcaacctgg
     1141 aggtgaagga gggagccccc ctgcccttct cctacgacat tctgaccacc gcgttcgcct
     1201 acggcaacag ggccttcacc aagtaccccg acgacatccc caactacttc aagcagtcct
     1261 tccccgaggg ctactcttgg gagcgcacca tgaccttcga ggacaagggc atcgtgaagg
     1321 tgaagtccga catctccatg gaggaggact ccttcatcta cgagatacac ctcaagggcg
     1381 agaacttccc ccccaacggc cccgtgatgc agaagaagac caccggctgg gacgcctcca
     1441 ccgagaggat gtacgtgcgc gacggcgtgc tgaagggcga cgtcaagcac aagctgctgc
     1501 tggagggcgg cggccaccac cgcgttgact tcaagaccat ctacagggcc aagaaggcgg
     1561 tgaagctgcc cgactatcac tttgtggacc accgcatcga gatcctgaac cacgacaagg
     1621 actacaacaa ggtgaccgtt tacgagagcg ccgtggcccg caactccacc gacggcatgg
     1681 acgagctgta caagagatct ggtaccatga ttgttttacc cagacgcctg tcagacgagg
     1741 ttgccgatcg tgtgcgggcg ctgattgatg aaaaaaacct ggaagcgggc atgaagttgc
     1801 ccgctgagcg ccaactggcg atgcaactcg gcgtatcacg taattcactg cgcgaggcgc
     1861 tggcaaaact ggtgagtgaa ggcgtgctgc tcagtcgacg cggcggcggg acgtttattc
     1921 gctggcgtca tgacacatgg tcggagcaaa acatcgtcca gccgctaaaa acactgatgg
     1981 ccgatgatcc ggattacagt ttcgatattc tggaagcccg ctacgccatt gaagccagca
     2041 ccgcatggca tgcggcaatg cgcgccacac ctggcgacaa agaaaagatt cagctttgct
     2101 ttgaagcaac gctaagtgaa gacccggata tcgcctcaca agcggacgtt cgttttgatc
     2161 tggcgattgc cgaagcctca cataacatcg tgctgctgca aaccatgcgc ggtttcttcg
     2221 atgtcctgca atcctcagtg aagcatagcc gtcagcggat gtatctggtg ccaccggttt
     2281 tttcacaact gaccgaacaa catcaggctg tcattgacgc catttttgcc ggtgatgctg
     2341 acggggcgcg taaagcaatg atggcgcacc ttagttttgt tcacaccacc atgaaacgat
     2401 tcgatgaaga tcaggctcgc cacgcacgga ttacccgcct gcccggtgag cataatgagc
     2461 attcgaggga gaaaaacgca cttaagagat ctaccatggt gagcaagggc gaggagctgt
     2521 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     2581 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagctgatct
     2641 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg ggctacggcc
     2701 tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     2761 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     2821 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     2881 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     2941 acaacgtcta tatcaccgcc gacaagcaga agaacggcat caaggccaac ttcaagatcc
     3001 gccacaacat cgaggacggc ggcgtgcagc tcgccgacca ctaccagcag aacaccccca
     3061 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagctaccag tccgccctga
     3121 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     3181 ggatcactct cggcatggac gagctgtaca agtaaaagct taagtttaaa ccgctgatca
     3241 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     3301 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     3361 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     3421 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     3481 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
     3541 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     3601 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     3661 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     3721 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     3781 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     3841 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     3901 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
     3961 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     4021 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     4081 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     4141 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     4201 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag
     4261 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg
     4321 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     4381 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     4441 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     4501 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt
     4561 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     4621 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     4681 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     4741 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     4801 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     4861 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg
     4921 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     4981 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     5041 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     5101 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     5161 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     5221 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     5281 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     5341 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     5401 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     5461 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     5521 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     5581 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5641 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5701 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5761 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5821 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5881 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5941 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     6001 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     6061 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     6121 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     6181 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     6241 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     6301 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     6361 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     6421 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     6481 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     6541 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     6601 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6661 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6721 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6781 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6841 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6901 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6961 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     7021 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     7081 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     7141 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     7201 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     7261 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     7321 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     7381 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     7441 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     7501 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     7561 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     7621 cacatttccc cgaaaagtgc cacctgacgt c
//
