LOCUS       sequence-407614-        6335 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of 2M-Qt-FLAG EMcapsulins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6335)
  AUTHORS   Sigmund F, Berezin O, Beliakova S, Magerl B, Drawitsch M, Piovesan 
            A, Goncalves F, Bodea SV, Winkler S, Bousraou Z, Grosshauser M, 
            Samara E, Pujol-Marti J, Schadler S, So C, Irsen S, Walch A, Kofler 
            F, Piraud M, Kornfeld J, Briggman K, Westmeyer GG
  TITLE     Genetically encoded barcodes for correlative volume electron 
            microscopy.
  JOURNAL   Nat Biotechnol. 2023 Apr 17. doi: 10.1038/s41587-023-01713-y.
  PUBMED    37069313
REFERENCE   2  (bases 1 to 6335)
  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: "Nat 
            Biotechnol. 2023 Apr 17. doi: 10.1038/s41587-023-01713-y."
COMMENT     Sequence Label: 2M-Qt-FLAG
FEATURES             Location/Qualifiers
     source          1..6335
                     /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"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      958..967
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2272..2295
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2343..2360)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2349..2573
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2619..3047
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2706..2725)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2916..2937
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3056..3076)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3061..3390
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3241..3376
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3303..3322
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3438..3485
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3504..3878
                     /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    4008..4129
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4045..4064)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4099..4118
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4178..4194)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4178..4194)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4191..4213)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4202..4218
                     /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(4226..4232,4233..4250,4251..4256))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4271..4292
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4409..4426)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4580..5168)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4660..4679)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5339..6130,6131..6199))
                     /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     5962..5981
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6200..6304)
                     /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 ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattcgcc
      961 accatggacc cagagacctg tccgtgtcca actggaggtt cttgtacgtg tagtgataag
     1021 tgtaaatgca agggatgtaa atgtactaac tgtaagaaat catgttgtag ttgttgccct
     1081 gccggatgtg agaaatgtgc gaaagactgt gtttgtaagg gggaagaggg tgccaaagct
     1141 gaggcggaaa aatgttcctg ttgtcaaggt ggaggttctg gcggtggatc gggaggttca
     1201 gcggatcctg aaacatgtcc ttgtcctact ggtggaagtt gtacttgttc tgataaatgt
     1261 aagtgcaaag gctgcaaatg taccaattgt aaaaaatctt gttgttcgtg ttgtcccgct
     1321 ggttgtgaaa agtgtgctaa agattgtgta tgtaaaggtg aagaaggagc aaaagcagaa
     1381 gccgagaagt gttcatgctg tcagggcagc ggcccgggca acaagagcca gctgtaccct
     1441 gacagccctc tgaccgacca ggacttcaac cagctggacc agaccgtgat tgaagccgcc
     1501 agaaggcagc tcgtcggcag aagattcatc gagctgtacg gccctctcgg cagaggcatg
     1561 cagagcgtgt tcaacgacat cttcatggaa agccacgagg ccaagatgga cttccagggc
     1621 agcttcgaca ccgaggtgga aagcagcaga agagtgaact acacaatccc catgctgtac
     1681 aaggacttcg tgctgtactg gcgcgacctg gaacagagca aggccctgga catccccatc
     1741 gatttttccg tggccgccaa cgccgctaga gatgtggcct ttctggaaga tcagatgatc
     1801 tttcacggca gcaaagagtt cgacatcccc ggcctgatga acgtgaaggg cagactgaca
     1861 cacctgatcg gcaattggta cgagagcggc aacgccttcc aggacatcgt ggaagcccgg
     1921 aacaagctgc tggaaatgaa ccacaacggc ccctacgctc tggtgctgag ccctgagctg
     1981 tatagcctgc tgcacagagt gcacaaggac accaacgtcc tggaaatcga gcacgtgcgc
     2041 gagctgatta ccgccggtgt ctttcagagc cctgtgctga agggaaagtc cggcgtgatc
     2101 gtgaacaccg gcagaaacaa cctggacctg gccatcagcg aggacttcga gacagcctat
     2161 ctgggcgaag agggcatgaa tcaccccttc cgggtgtacg agacagtggt gctgcggatt
     2221 aagaggcctg ccgccatctg caccctgatc gatcctgaag aaggctccgg cgactacaag
     2281 gacgacgacg acaaatgagc ggccgctcga gtctagaggg cccgtttaaa cccgctgatc
     2341 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2401 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2461 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2521 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2581 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     2641 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2701 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2761 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2821 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2881 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2941 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     3001 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     3061 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3121 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     3181 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3241 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3301 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     3361 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     3421 ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat
     3481 acgacaaggt gaggaactaa accatggcca agttgaccag tgccgttccg gtgctcaccg
     3541 cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact
     3601 tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg
     3661 tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg
     3721 agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg
     3781 ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg
     3841 gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctacga gatttcgatt
     3901 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     3961 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     4021 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4081 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     4141 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     4201 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4261 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4321 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4381 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4441 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4501 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4561 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4621 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4681 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4741 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4801 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4861 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4921 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4981 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5041 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5101 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5161 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5221 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5281 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5341 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5401 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5461 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5521 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5581 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5641 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5701 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5761 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5821 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5881 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5941 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6001 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6061 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6121 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6181 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6241 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6301 cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
