LOCUS       sequence-415626-        5324 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ER-targeted split-CFAST10 for Short-Range ER-Mito Contact 
            sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5324)
  TITLE     Mammalian expression plasmids for ER-Mitochondria contact sites 
            monitoring using splitFAST (OMM-SPLICS-NFAST and ER-SPLICS-CFAST10)
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5324)
  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: 
            "Unpublished"
COMMENT     Sequence Label: ER-SPLICSs-cFAST10
FEATURES             Location/Qualifiers
     source          1..5324
                     /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      935..944
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1235..1258
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1332..1349)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1338..1562
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1608..2036
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1695..1714)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1905..1926
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2045..2065)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2050..2379
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2230..2365
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2292..2311
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2427..2474
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2493..2867
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    2997..3118
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3034..3053)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3088..3107
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3167..3183)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3167..3183)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3180..3202)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3191..3207
                     /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(3215..3221,3222..3239,3240..3245))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3260..3281
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3398..3415)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3569..4157)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3649..3668)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4328..5119,5120..5188))
                     /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     4951..4970
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5189..5293)
                     /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 gtttaaactt aagcttggta ccgagctcgg atccgccgcc atgggtgaca gctattgggt
      961 ctttgtgaaa cgggaattcg gcggagatgg cggatctggc ggcggagata tcagcaagct
     1021 gagagtgttc ctggccctgc ccatcatcat ggtggtggcc ttttctatgt gcatcatctg
     1081 tctgctgatg gcaggcgaca cctggacaga gaccctggcc tacgtgctgt tttggggcgt
     1141 ggccagcatc ggcacattct ttatcatcct gtataacggc aaggacttcg tggacgcccc
     1201 cagactggtg cagaaagaga agatcgacga tatcgattac aaggatgacg acgataagtg
     1261 atctagaccg tcgacacgga attcattgcg gccgctcgag tctagagggc ccgtttaaac
     1321 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     1381 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     1441 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     1501 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     1561 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     1621 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     1681 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     1741 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     1801 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     1861 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     1921 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     1981 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     2041 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     2101 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     2161 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     2221 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     2281 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     2341 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     2401 tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatag tatatcggca
     2461 tagtataata cgacaaggtg aggaactaaa ccatggccaa gttgaccagt gccgttccgg
     2521 tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct
     2581 cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca
     2641 tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg
     2701 gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct
     2761 ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg
     2821 acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgacac gtgctacgag
     2881 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     2941 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     3001 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     3061 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     3121 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     3181 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3241 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3301 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     3361 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3421 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3481 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3541 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     3601 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3661 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3721 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     3781 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     3841 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3901 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     3961 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4021 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4081 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4141 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4201 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4261 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4321 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4381 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4441 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4501 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4561 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     4621 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     4681 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     4741 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     4801 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     4861 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     4921 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     4981 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5041 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5101 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5161 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5221 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5281 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
