LOCUS       sequence-491013-        6285 bp    DNA     circular SYN 29-OCT-2025
DEFINITION  anchoring mCherry-1x ALFAto the chromatin/nucleus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6285)
  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 6285)
  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: H2B_mCherry_ALFA
FEATURES             Location/Qualifiers
     source          1..6285
                     /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             1012..1389
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     CDS             1402..2106
                     /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(1544..1562)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1812..1831
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2182..2220
                     /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(2293..2310)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2299..2523
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2569..2997
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2656..2675)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2866..2887
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3006..3026)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3011..3340
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3191..3326
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3253..3272
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3388..3435
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3454..3828
                     /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    3958..4079
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3995..4014)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4049..4068
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4128..4144)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4128..4144)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4141..4163)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4152..4168
                     /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(4176..4182,4183..4200,4201..4206))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4221..4242
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4359..4376)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4530..5118)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4610..4629)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5289..6080,6081..6149))
                     /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     5912..5931
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6150..6254)
                     /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 gtaccgccac catgccagag
     1021 ccagcgaagt ctgctcccgc cccgaaaaag ggctccaaga aggcggtgac taaggcgcag
     1081 aagaaaggcg gcaagaagcg caagcgcagc cgcaaggaga gctattccat ctatgtgtac
     1141 aaggttctga agcaggtcca ccctgacacc ggcatttcgt ccaaggccat gggcatcatg
     1201 aattcgtttg tgaacgacat tttcgagcgc atcgcaggtg aggcttcccg cctggcgcat
     1261 tacaacaagc gctcgaccat cacctccagg gagatccaga cggccgtgcg cctgctgctg
     1321 cctggggagt tggccaagca cgccgtgtcc gagggtacta aggccatcac caagtacacc
     1381 agcgctaagg atcaattcgg tgtgagcaag ggcgaggagg ataacatggc catcatcaag
     1441 gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc
     1501 gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc
     1561 aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc
     1621 aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag
     1681 ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag
     1741 gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc
     1801 ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg
     1861 atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac
     1921 ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg
     1981 cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc
     2041 atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg
     2101 tacaagggtg gtggtggcgc ggccgcagat cgatcgatca cgcgtgccac catgactagt
     2161 tttgtacaaa gcggtccgcc atcacgtttg gaagaggaac tgagacgccg cttaactgaa
     2221 cctggccgcg gtaaacatat ggatcgatca taatactcga gtctagaggg cccgtttaaa
     2281 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2341 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2401 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2461 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     2521 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     2581 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2641 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2701 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2761 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2821 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2881 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     2941 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     3001 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     3061 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     3121 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3181 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3241 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     3301 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     3361 atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc
     3421 atagtataat acgacaaggt gaggaactaa accatggcca agttgaccag tgccgttccg
     3481 gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc
     3541 tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc
     3601 atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc
     3661 ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc
     3721 tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc
     3781 gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctacga
     3841 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     3901 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     3961 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4021 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     4081 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     4141 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     4201 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     4261 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4321 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4381 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4441 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4501 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4561 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4621 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4681 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4741 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4801 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4861 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4921 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     4981 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5041 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5101 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5161 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     5221 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5281 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5341 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5401 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5461 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5521 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5581 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5641 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5701 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5761 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5821 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5881 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5941 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6001 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6061 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6121 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     6181 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6241 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
