LOCUS       sequence-425794-        7374 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 7374)
  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 7374)
  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..7374
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    930..1051
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(967..986)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1153..1169)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1153..1169)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1166..1188)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1177..1193
                     /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(1201..1207,1208..1225,1226..1231))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1246..1267
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1384..1401)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1555..2143)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1635..1654)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2314..3105,3106..3174))
                     /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     2937..2956
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3175..3279)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3354..3373)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3545..3924
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3925..4128
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4079..4099
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4173..4192
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4173..4191
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4241..4250
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4640..4720
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             4739..4780
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     intron          5032..5261
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     5244..5263
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(5333..5907,5908..5910,5911..5919)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5500..5517)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5727..5746
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      6251..6260
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6257..7147
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
ORIGIN
        1 atattacttc agctatccct tgttcgtggg tttcgcgcta cttcggctgg tcgcgtttca
       61 tctaggtttg ttgtttgtct ggttatgtca acggttctcg cgagcgttaa tggcagcaaa
      121 gaggtcggca aatagcgaat cgcctcataa acgcaacctc tattacttct cttacccgtt
      181 gttcgttggc ttcgctctcc ttcggctagt tgcgtttcat cttggtcttc tgttcgtgtg
      241 gctttgccaa agattttcgc gagctttaat ggcggctaag aggagtgcca atagcgaatc
      301 cccgcataag cggaacttat actacttctc ttaccctctt tttgtaggct ttgctctact
      361 ccggctagtg gccttccatc taggcctact attcgtctgg ctctgccagc gcttctcgcg
      421 agccttgatg gcagctaagc gttccgcaaa tagcgaatca cctcataagc ggaatctgta
      481 ctatttctca tatcctctct tcgtagggtt cgcactatta cgtcttgtag catttcatct
      541 gggattatta ttcgtctggt tgtgccagcg attttcacga gccttaatgg ctgcgaagag
      601 aagcgcaaat tctgaatctc ctcacaagcg gaacctctat tactttagtt acccgctctt
      661 cgtcggtttc gcgttactca ggctggttgc attccacctc ggcttgctgt tcgtatggct
      721 ttgccagaga ttctcccggg cgctaatggc agccaagcgc tcctaaggat ccaccggatc
      781 ggtacaagta actcgagggc caattaatta actctagata actgatcata atcagccata
      841 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
      901 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
      961 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     1021 gtggtttgtc caaactcatc aatgtatctt aacgcgtcga gtgcattcta gttgtggttt
     1081 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
     1141 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     1201 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     1261 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     1321 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     1381 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     1441 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     1501 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     1561 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     1621 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     1681 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     1741 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     1801 gggctgtgtg cgcgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     1861 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     1921 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     1981 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     2041 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     2101 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     2161 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     2221 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     2281 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     2341 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     2401 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     2461 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     2521 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     2581 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     2641 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     2701 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     2761 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     2821 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     2881 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     2941 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     3001 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     3061 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     3121 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     3181 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     3241 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     3301 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
     3361 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
     3421 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
     3481 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
     3541 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
     3601 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     3661 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     3721 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     3781 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     3841 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     3901 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
     3961 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     4021 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     4081 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
     4141 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
     4201 gcttggtacc gagctcggat cgatatctgc ggcctagcta gccaccatgg agaccgtgag
     4261 attcggcggc gacgacatcg agaacagcct ggccaagatg gacgacaagg ccctggacaa
     4321 gctggccttc ggcgccatcc agctggacgg caacggcaag atcatccact acaacgccgc
     4381 cgagggcacc atcaccggca gagaccccaa gaccgtgatc ggcaagaact tcttcaccga
     4441 cgtggccccc ggcacccaga gcaaggagtt ccagggcaga ttcaaggagg gcgtgcagaa
     4501 gggcgacctg aacaccatgt tcgagtggat gatccccacc agcagaggcc ccaccaaggt
     4561 gaaggtgcac atgaagaagg ccatgacctc cggaggaggc ggcagcggcg gagggggatc
     4621 accggtcgcc ggtagcggtt acccatacga tgttccagat tacgcttatc cttatgacgt
     4681 acctgactat gcataccctt atgatgtacc agactacgct tcaggaggtg gagggagtgg
     4741 caagcccatc cccaaccccc tgctgggcct ggacagcacc aaacttggta gtgggagcgg
     4801 ctccggagct tcgaattctt tgcaggtgca gaatggacct ataggacatc gaggtgaagg
     4861 ggacccaagt ggtattccca tatttatggt gctggtgcca gtgtttgccc tcaccatggt
     4921 agcagcctgg gctttcatga gataccggca acaactttga taagcggccg catagataac
     4981 tgatccagtg tgctggaatt aattcgctgt ctgcgagggc cagctgttgg ggtgagtact
     5041 ccctctcaaa agcgggcatg acttctgcgc taagattgtc agtttccaaa aacgaggagg
     5101 atttgatatt cacctggccc gcggtgatgc ctttgagggt ggccgcgtcc atctggtcag
     5161 aaaagacaat ctttttgttg tcaagcttga ggtgtggcag gcttgagatc tggccataca
     5221 cttgagtgac aatgacatcc actttgcctt tctctccaca ggtgtccact cccaggtcca
     5281 actgcaggtc gagcatgcat ctagggcggc caattccgcc cctctccccc cccccctctc
     5341 cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg
     5401 tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg
     5461 gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag
     5521 gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt
     5581 ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc
     5641 aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga
     5701 gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga
     5761 aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct
     5821 ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg
     5881 ttttcctttg aaaaacacga tgataagctt gccacaaccc gggaggcgcg ccatgggtga
     5941 cagctattgg gtctttgtga aacgggtgtc cactagtgaa accgaaaccg gaggcggctc
     6001 cggaggcagc ggcggttctg gaggttccac cctaccggtc tcagctggag gcagtgctgg
     6061 tggaagtgcc ggaggtagaa ttctagaagg tgccgcaaga gaggctgccg ctagggaagc
     6121 agctgcacgt gaggcagctg ctagagaagc tgcagccagg gaggctgcag ctagagcatc
     6181 aggtggtggc ggttcaggcg gaggtggatc tggaggtggc ggatcgggtg ggggaggttc
     6241 gacaccggtc gccaccatgg cagaaatcgg tactggcttt ccattcgacc cccattatgt
     6301 ggaagtcctg ggcgagcgca tgcactacgt cgatgttggt ccgcgcgatg gcacccctgt
     6361 gctgttcctg cacggtaacc cgacctcctc ctacgtgtgg cgcaacatca tcccgcatgt
     6421 tgcaccgacc catcgctgca ttgctccaga cctgatcggt atgggcaaat ccgacaaacc
     6481 agacctgggt tatttcttcg acgaccacgt ccgcttcatg gatgccttca tcgaagccct
     6541 gggtctggaa gaggtcgtcc tggtcattca cgactggggc tccgctctgg gtttccactg
     6601 ggccaagcgc aatccagagc gcgtcaaagg tattgcattt atggagttca tccgccctat
     6661 cccgacctgg gacgaatggc cagaatttgc ccgcgagacc ttccaggcct tccgcaccac
     6721 cgacgtcggc cgcaagctga tcatcgatca gaacgttttt atcgagggta cgctgccgat
     6781 gggtgtcgtc cgcccgctga ctgaagtcga gatggaccat taccgcgagc cgttcctgaa
     6841 tcctgttgac cgcgagccac tgtggcgctt cccaaacgag ctgccaatcg ccggtgagcc
     6901 agcgaacatc gtcgcgctgg tcgaagaata catggactgg ctgcaccagt cccctgtccc
     6961 gaagctgctg ttctggggca ccccaggcgt tctgatccca ccggccgaag ccgctcgcct
     7021 ggccaaaagc ctgcctaact gcaaggctgt ggacatcggc ccgggtctga atctgctgca
     7081 agaagacaac ccggacctga tcggcagcga gatcgcgcgc tggctgtcga cgctcgagat
     7141 ttccggcatg tacaagtccg gactcagatc tcgagctcaa gcatcgaatt ccgaatcccc
     7201 gcacaaacgc aacctgtact acttctctta cccccttttt gtcgggtttg ctctacttcg
     7261 attggttgct tttcatcttg gattactgtt tgtttggttg tgtcaacgct tttctagagc
     7321 gctgatggct gcgaaacgtt cggctaatag tgaatcgccc cataagagga actt
//
