LOCUS       sequence-491039-        7292 bp    DNA     circular SYN 29-OCT-2025
DEFINITION  anchoring mCherry-1x ALFA to the intermediate filaments.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7292)
  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 7292)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 29, 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: Vimentin_mCherry_ALFA
FEATURES             Location/Qualifiers
     source          1..7292
                     /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             2409..3113
                     /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(2551..2569)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2819..2838
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3189..3227
                     /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(3300..3317)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3306..3530
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3576..4004
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3663..3682)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3873..3894
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4013..4033)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4018..4347
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4198..4333
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        4395..4442
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4461..4835
                     /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    4965..5086
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5002..5021)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5056..5075
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5135..5151)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5135..5151)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5148..5170)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5159..5175
                     /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(5183..5189,5190..5207,5208..5213))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5228..5249
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5366..5383)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5537..6125)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5617..5636)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6296..7087,7088..7156))
                     /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     6919..6938
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7157..7261)
                     /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 catgtctacc aggtctgtgt
     1021 cctcgtcctc ctaccgcagg atgttcggtg gctccggcac atcgagccgg cccagctcca
     1081 accggagcta tgtgaccacg tccacacgca cctacagtct gggcagcgca ctgcgcccca
     1141 gcactagccg cagcctctat tcctcatccc ccggtggcgc ctatgtgacc cggtcctcgg
     1201 cagtgcgcct gcggagcagc gtgccgggcg tgcggctgct tcaagactcg gtggacttct
     1261 cgctggccga cgccatcaac actgagttca agaacacccg caccaacgag aaggtagaac
     1321 tgcaggagct gaatgaccgc tttgccaact acatcgacaa ggtgcgcttc ctcgagcagc
     1381 agaacaaaat cctgctggct gagctcgagc agctcaaggg ccagggcaag tcgcgcctgg
     1441 gcgacctgta cgaggaggag atgcgggagc tgcgccggca ggtggatcag ctcaccaacg
     1501 acaaggcccg tgtcgaggtg gagcgggaca acctggccga ggacatcatg cggctgcgag
     1561 agaaattgca ggaggagatg ctccagagag aggaagccga aagcaccctg cagtcattca
     1621 gacaggatgt tgacaatgct tctctggcac gtcttgacct tgaacggaaa gtggaatcct
     1681 tgcaggaaga aattgccttt ttgaagaaac tgcacgatga agagatccag gagctgcagg
     1741 cccagattca ggaacagcat gtccagatcg atgtggacgt ttccaagcct gacctcactg
     1801 ctgccctgcg tgatgtgcgc cagcagtatg aaagcgtggc tgccaagaac ctccaggagg
     1861 ccgaggaatg gtacaagtcc aagtttgctg acctctctga ggctgccaac cggaacaacg
     1921 atgccctgcg ccaggccaag caggagtcaa acgagtaccg gagacaggtg cagtcactca
     1981 cctgtgaagt ggatgccctt aaaggcacta acgagtccct ggagcgccag atgcgtgaga
     2041 tggaagagaa ttttgccctt gaagctgcta actaccagga cactattggc cgcctgcagg
     2101 atgagatcca aaacatgaag gaagagatgg ctcgtcacct tcgtgaatac caagatctgc
     2161 tcaatgttaa gatggccctg gacattgaga tcgccaccta caggaagctg ctggaaggcg
     2221 aggagagcag gatttctctg cctctgccaa ccttttcttc cctgaacctg agagaaacta
     2281 acctggagtc acttcctctg gttgacaccc actcaaaaag aacactcctg attaagacgg
     2341 ttgagaccag agatggacag gtgatcaatg agacttctca gcatcacgat gaccttgaag
     2401 aattcggtgt gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct
     2461 tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg
     2521 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc
     2581 tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga
     2641 agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg
     2701 agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc
     2761 aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc
     2821 ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg
     2881 acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg
     2941 acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca
     3001 acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt
     3061 acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagggtggtg
     3121 gtggcgcggc cgcagatcga tcgatcacgc gtgccaccat gactagtttt gtacaaagcg
     3181 gtccgccatc acgtttggaa gaggaactga gacgccgctt aactgaacct ggccgcggta
     3241 aacatatgga tcgatcataa tactcgagtc tagagggccc gtttaaaccc gctgatcagc
     3301 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     3361 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     3421 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     3481 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     3541 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     3601 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     3661 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3721 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3781 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     3841 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     3901 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     3961 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     4021 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     4081 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     4141 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     4201 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     4261 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     4321 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     4381 tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg
     4441 acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc
     4501 gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg
     4561 tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc
     4621 aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc
     4681 tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca
     4741 tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca
     4801 actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctacgagat ttcgattcca
     4861 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4921 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4981 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     5041 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     5101 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     5161 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5221 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5281 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5341 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5401 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5461 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5521 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5581 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5641 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5701 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5761 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5821 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5881 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5941 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     6001 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6061 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6121 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6181 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6241 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6301 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6361 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6421 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6481 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6541 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6601 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6661 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6721 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6781 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6841 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6901 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6961 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     7021 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7081 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7141 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7201 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7261 gcacatttcc ccgaaaagtg ccacctgacg tc
//
