LOCUS       sequence-418333-        7360 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Detection of mito-lipid droplet contact site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7360)
  AUTHORS   Li X, Gamuyao R, Wu ML, Cho WJ, King SV, Petersen RA, Stabley DR, 
            Lindow C, Climer LK, Shirinifard A, Ferrara F, Throm RE, Robinson 
            CG, Zhou Y, Carisey AF, Tebo AG, Chang CL
  TITLE     A fluorogenic complementation tool kit for interrogating lipid 
            droplet-organelle interaction.
  JOURNAL   J Cell Biol. 2024 Sep 2;223(9):e202311126. doi: 
            10.1083/jcb.202311126. Epub 2024 Jul 1.
  PUBMED    38949658
REFERENCE   2  (bases 1 to 7360)
  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: "J Cell 
            Biol."; date: "2024-09-2"; volume: "223(9):e202311126. doi"; pages: 
            " 10.1083/jcb.202311126. Epub 2024 Jul 1"
COMMENT     Sequence Label: FAB(mito-LD)
FEATURES             Location/Qualifiers
     source          1..7360
                     /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"
     regulatory      931..940
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1330..1410
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             1429..1470
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     intron          1722..1951
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1934..1953
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(2009..2583,2584..2586,2587..2595)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2176..2193)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2403..2422
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      2927..2936
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2933..3823
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     polyA_signal    4980..5101
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5017..5036)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5203..5219)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5203..5219)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5216..5238)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5227..5243
                     /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(5251..5257,5258..5275,5276..5281))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5296..5317
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5434..5451)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5605..6193)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5685..5704)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6364..7155,7156..7224))
                     /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     6987..7006
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7225..7329)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct 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 atgggtggac 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 gcttggtacc
      901 gagctcggat cgatatctgc ggcctagcta gccaccatgg agaccgtgag attcggcggc
      961 gacgacatcg agaacagcct ggccaagatg gacgacaagg ccctggacaa gctggccttc
     1021 ggcgccatcc agctggacgg caacggcaag atcatccact acaacgccgc cgagggcacc
     1081 atcaccggca gagaccccaa gaccgtgatc ggcaagaact tcttcaccga cgtggccccc
     1141 ggcacccaga gcaaggagtt ccagggcaga ttcaaggagg gcgtgcagaa gggcgacctg
     1201 aacaccatgt tcgagtggat gatccccacc agcagaggcc ccaccaaggt gaaggtgcac
     1261 atgaagaagg ccatgacctc cggaggaggc ggcagcggcg gagggggatc accggtcgcc
     1321 ggtagcggtt acccatacga tgttccagat tacgcttatc cttatgacgt acctgactat
     1381 gcataccctt atgatgtacc agactacgct tcaggaggtg gagggagtgg caagcccatc
     1441 cccaaccccc tgctgggcct ggacagcacc aaacttggta gtgggagcgg ctccggagct
     1501 tcgaattctt tgcaggtgca gaatggacct ataggacatc gaggtgaagg ggacccaagt
     1561 ggtattccca tatttatggt gctggtgcca gtgtttgccc tcaccatggt agcagcctgg
     1621 gctttcatga gataccggca acaactttga taagcggccg catagataac tgatccagtg
     1681 tgctggaatt aattcgctgt ctgcgagggc cagctgttgg ggtgagtact ccctctcaaa
     1741 agcgggcatg acttctgcgc taagattgtc agtttccaaa aacgaggagg atttgatatt
     1801 cacctggccc gcggtgatgc ctttgagggt ggccgcgtcc atctggtcag aaaagacaat
     1861 ctttttgttg tcaagcttga ggtgtggcag gcttgagatc tggccataca cttgagtgac
     1921 aatgacatcc actttgcctt tctctccaca ggtgtccact cccaggtcca actgcaggtc
     1981 gagcatgcat ctagggcggc caattccgcc cctctccctc ccccccccct aacgttactg
     2041 gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat
     2101 tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc
     2161 ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag
     2221 cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc
     2281 ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac
     2341 ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca
     2401 aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt
     2461 gtatgggatc tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa
     2521 aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgatgat
     2581 aagcttgcca caacccggga ggcgcgccat gggtgacagc tattgggtct ttgtgaaacg
     2641 ggtgtccact agtgaaaccg aaaccggagg cggctccgga ggcagcggcg gttctggagg
     2701 ttccacccta ccggtctcag ctggaggcag tgcaggtgga tctgccggag gtagaattct
     2761 agaagctgcc gcaagagagg cagccgctag ggaagcagct gcacgtgagg cagctgctag
     2821 agaagctgca gccagggagg ctgcagctag agcatcaggt ggtggcggtt caggcggagg
     2881 tggctctggc ggtggcggat cgggtggcgg aggttcgaca ccggtcgcca ccatggcaga
     2941 aatcggtact ggctttccat tcgaccccca ttatgtggaa gtcctgggcg agcgcatgca
     3001 ctacgtcgat gttggtccgc gcgatggcac ccctgtgctg ttcctgcacg gtaacccgac
     3061 ctcctcctac gtgtggcgca acatcatccc gcatgttgca ccgacccatc gctgcattgc
     3121 tccagacctg atcggtatgg gcaaatccga caaaccagac ctgggttatt tcttcgacga
     3181 ccacgtccgc ttcatggatg ccttcatcga agccctgggt ctggaagagg tcgtcctggt
     3241 cattcacgac tggggctccg ctctgggttt ccactgggcc aagcgcaatc cagagcgcgt
     3301 caaaggtatt gcatttatgg agttcatccg ccctatcccg acctgggacg aatggccaga
     3361 atttgcccgc gagaccttcc aggccttccg caccaccgac gtcggccgca agctgatcat
     3421 cgatcagaac gtttttatcg agggtacgct gccgatgggt gtcgtccgcc cgctgactga
     3481 agtcgagatg gaccattacc gcgagccgtt cctgaatcct gttgaccgcg agccactgtg
     3541 gcgcttccca aacgagctgc caatcgccgg tgagccagcg aacatcgtcg cgctggtcga
     3601 agaatacatg gactggctgc accagtcccc tgtcccgaag ctgctgttct ggggcacccc
     3661 aggcgttctg atcccaccgg ccgaagccgc tcgcctggcc aaaagcctgc ctaactgcaa
     3721 ggctgtggac atcggcccgg gtctgaatct gctgcaagaa gacaacccgg acctgatcgg
     3781 cagcgagatc gcgcgctggc tgtcgacgct cgagatttcc ggcatgtaca agtccggact
     3841 cagatctcga gctcaagcat cgaattccga atccccgcac aaacgcaacc tgtactactt
     3901 ctcttacccc ctttttgtcg ggtttgctct acttcgattg gttgcttttc atcttggatt
     3961 actgtttgtt tggttgtgtc aacgcttttc tagagcgctg atggctgcga aacgttcggc
     4021 taatagtgaa tcgccccata agaggaactt atattacttc agctatccct tgttcgtggg
     4081 tttcgcgcta cttcggctgg tcgcgtttca tctaggtttg ttgtttgtct ggttatgtca
     4141 acggttctcg cgagcgttaa tggcagcaaa gaggtcggca aatagcgaat cgcctcataa
     4201 acgcaacctc tattacttct cttacccgtt gttcgttggc ttcgctctcc ttcggctagt
     4261 tgcgtttcat cttggtcttc tgttcgtgtg gctttgccaa agattttcgc gagctttaat
     4321 ggcggctaag aggagtgcca atagcgaatc cccgcataag cggaacttat actacttctc
     4381 ttaccctctt tttgtaggct ttgctctact ccggctagtg gccttccatc taggcctact
     4441 attcgtctgg ctctgccagc gcttctcgcg agccttgatg gcagctaagc gttccgcaaa
     4501 tagcgaatca cctcataagc ggaatctgta ctatttctca tatcctctct tcgtagggtt
     4561 cgcactatta cgtcttgtag catttcatct gggattatta ttcgtctggt tgtgccagcg
     4621 attttcacga gccttaatgg ctgcgaagag aagcgcaaat tctgaatctc ctcacaagcg
     4681 gaacctctat tactttagtt acccgctctt cgtcggtttc gcgttactca ggctggttgc
     4741 attccacctc ggcttgctgt tcgtatggct ttgccagaga ttctcccggg cgctaatggc
     4801 agccaagcgc tcctaaggat ccaccggatc ggtacaagta actcgagggc caattaatta
     4861 actctagata actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa
     4921 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
     4981 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     5041 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     5101 aacgcgtcga gtgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     5161 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     5221 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5281 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5341 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5401 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5461 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5521 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5581 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5641 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5701 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5761 gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct
     5821 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5881 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5941 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     6001 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     6061 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6121 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     6181 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     6241 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     6301 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6361 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6421 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6481 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     6541 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6601 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6661 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6721 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6781 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6841 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6901 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6961 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     7021 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     7081 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     7141 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     7201 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     7261 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     7321 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
