LOCUS       sequence-491012-        6384 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  anchoring 1xHA-mCherry to the chromatin/nucleus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6384)
  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 6384)
  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: 
            "Development."; date: "2021-03-16"; volume: "148(6):dev191700. doi";
            pages: " 10.1242/dev.191700"
COMMENT     Sequence Label: HA_mCherry_H2B
FEATURES             Location/Qualifiers
     source          1..6384
                     /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             964..990
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      997..1006
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1003..1710
                     /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(1148..1166)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1416..1435
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1717..2097
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     primer_bind     complement(2135..2152)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2141..2365
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2411..2839
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2498..2517)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2708..2729
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2848..2868)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2853..3182
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3033..3168
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3095..3114
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3245..3844
                     /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(3245..3264)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3741..3761
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4057..4178
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4094..4113)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4148..4167
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4227..4243)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4227..4243)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4240..4262)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4251..4267
                     /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(4275..4281,4282..4299,4300..4305))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4320..4341
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4458..4475)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4629..5217)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4709..4728)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5388..6179,6180..6248))
                     /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     6011..6030
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6249..6353)
                     /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 atgtacccat acgatgttcc agattacgct aagctcgcca ccatggtgag caagggcgag
     1021 gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc
     1081 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
     1141 cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg
     1201 tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac
     1261 tacttgaagc tgtccttctc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
     1321 ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag
     1381 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg
     1441 ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc
     1501 aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac
     1561 aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc
     1621 acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac
     1681 tccaccggcg gcatggacga gctgtacaag gatatcatgc cagagccagc gaagtctgct
     1741 cccgccccga aaaagggctc caagaaggcg gtgactaagg cgcagaagaa aggcggcaag
     1801 aagcgcaagc gcagccgcaa ggagagctat tccatctatg tgtacaaggt tctgaagcag
     1861 gtccaccctg acaccggcat ttcgtccaag gccatgggca tcatgaattc gtttgtgaac
     1921 gacattttcg agcgcatcgc aggtgaggct tcccgcctgg cgcattacaa caagcgctcg
     1981 accatcacct ccagggagat ccagacggcc gtgcgcctgc tgctgcctgg ggagttggcc
     2041 aagcacgccg tgtccgaggg tactaaggcc atcaccaagt acaccagcgc taagtgactc
     2101 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     2161 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     2221 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     2281 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     2341 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     2401 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     2461 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     2521 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     2581 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     2641 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     2701 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     2761 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     2821 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     2881 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     2941 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3001 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3061 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3121 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3181 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaagct
     3241 taccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc
     3301 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgaccc
     3361 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct
     3421 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc
     3481 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag
     3541 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa
     3601 ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct
     3661 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
     3721 cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac
     3781 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
     3841 ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac cccatgcatc
     3901 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     3961 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     4021 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     4081 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     4141 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     4201 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     4261 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     4321 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4381 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4441 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4501 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4561 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4621 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4681 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4741 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4801 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4861 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4921 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4981 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5041 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     5101 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5161 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5221 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5281 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     5341 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5401 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     5461 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5521 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5581 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5641 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5701 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5761 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5821 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5881 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5941 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6001 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6061 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6121 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6181 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6241 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6301 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6361 ccccgaaaag tgccacctga cgtc
//
