LOCUS       sequence-491007-        6434 bp    DNA     circular SYN 29-OCT-2025
DEFINITION  anchoring 1xALFA-mCherry to the chromatin/nucleus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6434)
  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 6434)
  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: ALFA_mCherry_H2B
FEATURES             Location/Qualifiers
     source          1..6434
                     /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             996..1034
                     /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"
     regulatory      1047..1056
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1053..1760
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFSEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1198..1216)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1466..1485
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1767..2147
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     primer_bind     complement(2185..2202)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2191..2415
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2461..2889
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2548..2567)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2758..2779
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2898..2918)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2903..3232
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3083..3218
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3145..3164
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3295..3894
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3295..3314)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3791..3811
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4107..4228
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4144..4163)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4198..4217
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4277..4293)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4277..4293)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4290..4312)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4301..4317
                     /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(4325..4331,4332..4349,4350..4355))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4370..4391
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4508..4525)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4679..5267)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4759..4778)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5438..6229,6230..6298))
                     /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     6061..6080
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6299..6403)
                     /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 cttatcgcaa tgccatcacg tttggaagag gaactgagac
     1021 gccgcttaac tgaacctggc cggatcgcca ccatggtgag caagggcgag gaggataaca
     1081 tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc
     1141 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
     1201 agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt
     1261 tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc
     1321 tgtccttctc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
     1381 tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc
     1441 gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     1501 cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc
     1561 tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga
     1621 agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca
     1681 acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg
     1741 gcatggacga gctgtacaag gatatcatgc cagagccagc gaagtctgct cccgccccga
     1801 aaaagggctc caagaaggcg gtgactaagg cgcagaagaa aggcggcaag aagcgcaagc
     1861 gcagccgcaa ggagagctat tccatctatg tgtacaaggt tctgaagcag gtccaccctg
     1921 acaccggcat ttcgtccaag gccatgggca tcatgaattc gtttgtgaac gacattttcg
     1981 agcgcatcgc aggtgaggct tcccgcctgg cgcattacaa caagcgctcg accatcacct
     2041 ccagggagat ccagacggcc gtgcgcctgc tgctgcctgg ggagttggcc aagcacgccg
     2101 tgtccgaggg tactaaggcc atcaccaagt acaccagcgc taagtgactc gagtctagag
     2161 ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     2221 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     2281 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     2341 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     2401 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     2461 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2521 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     2581 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     2641 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     2701 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     2761 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     2821 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     2881 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     2941 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     3001 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     3061 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     3121 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     3181 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     3241 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaagct taccatgacc
     3301 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     3361 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgaccc ggaccgccac
     3421 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     3481 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     3541 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     3601 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     3661 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     3721 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
     3781 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     3841 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacgcccg
     3901 ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac cccatgcatc gaaatgaccg
     3961 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     4021 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     4081 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     4141 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4201 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     4261 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     4321 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     4381 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4441 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     4501 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4561 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4621 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4681 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4741 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4801 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4861 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4921 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4981 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5041 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5101 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5161 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5221 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5281 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5341 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5401 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5461 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5521 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5581 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5641 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5701 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5761 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5821 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5881 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5941 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     6001 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     6061 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     6121 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     6181 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6241 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     6301 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6361 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6421 tgccacctga cgtc
//
