LOCUS       sequence-490932-        6095 bp    DNA     circular SYN 28-OCT-2025
DEFINITION  anchoring mCherry-1x ALFA to the mitochondrial membrane.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6095)
  AUTHORS   Vigano MA, Ell CM, Kustermann MMM, Aguilar G, Matsuda S, Zhao N, 
            Stasevich TJ, Affolter M, Pyrowolakis G
  TITLE     Protein manipulation using single copies of short peptide tags in 
            cultured cells and in Drosophila melanogaster.
  JOURNAL   Development. 2021 Mar 16;148(6):dev191700. doi: 10.1242/dev.191700.
  PUBMED    33593816
REFERENCE   2  (bases 1 to 6095)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 28, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Development."; date: "2021-03-16"; volume: "148(6):dev191700. doi";
            pages: " 10.1242/dev.191700"
COMMENT     Sequence Label: mito_mCherry_ALFA
FEATURES             Location/Qualifiers
     source          1..6095
                     /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"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1212..1916
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1354..1372)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1622..1641
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1992..2030
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     primer_bind     complement(2103..2120)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2109..2333
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2379..2807
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2466..2485)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2676..2697
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2816..2836)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2821..3150
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3001..3136
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3063..3082
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3198..3245
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3264..3638
                     /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    3768..3889
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3805..3824)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3859..3878
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3938..3954)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3938..3954)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3951..3973)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3962..3978
                     /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(3986..3992,3993..4010,4011..4016))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4031..4052
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4169..4186)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4340..4928)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4420..4439)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5099..5890,5891..5959))
                     /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     5722..5741
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5960..6064)
                     /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 ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtaccg agctcggatc cgccaccatg agtctgactt
     1021 ccagttccag cgtacgagtt gaatggatcg cagcagttac cattgctgct gggacagctg
     1081 caattggtta tctagcttac aaaagatttt atgttaaaga tcatcgaaat aaagctatga
     1141 taaaccttca catccagaaa gacaacccca agatagtaca tgctggaagc ttaccggtcg
     1201 ccgaattcgg tgtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc
     1261 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
     1321 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
     1381 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg
     1441 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt
     1501 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
     1561 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
     1621 gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg
     1681 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact
     1741 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct
     1801 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac
     1861 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagggtg
     1921 gtggtggcgc ggccgcagat cgatcgatca cgcgtgccac catgactagt tttgtacaaa
     1981 gcggtccgcc atcacgtttg gaagaggaac tgagacgccg cttaactgaa cctggccgcg
     2041 gtaaacatat ggatcgatca taatactcga gtctagaggg cccgtttaaa cccgctgatc
     2101 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2161 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2221 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2281 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2341 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     2401 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2461 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2521 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2581 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2641 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2701 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     2761 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     2821 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     2881 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     2941 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3001 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3061 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     3121 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     3181 ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat
     3241 acgacaaggt gaggaactaa accatggcca agttgaccag tgccgttccg gtgctcaccg
     3301 cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact
     3361 tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg
     3421 tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg
     3481 agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg
     3541 ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg
     3601 gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctacga gatttcgatt
     3661 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     3721 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     3781 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3841 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     3901 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     3961 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4021 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4081 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4141 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4201 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4261 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4321 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4381 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4441 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4501 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4561 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4621 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4681 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4741 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4801 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4861 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4921 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4981 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5041 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5101 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5161 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5221 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5281 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5341 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5401 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5461 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5521 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5581 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5641 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5701 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5761 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5821 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5881 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5941 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6001 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6061 cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
