LOCUS       sequence-418121-        6350 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of 2M-Mx-FLAG EMcapsulins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6350)
  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 6350)
  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-Mx-FLAG
FEATURES             Location/Qualifiers
     source          1..6350
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(52..71)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        243..622
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        623..826
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     777..797
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     871..890
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        871..889
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      966..975
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2295..2318
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2366..2383)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2372..2596
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2642..3070
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2729..2748)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2939..2960
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3079..3099)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3084..3413
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3264..3399
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3326..3345
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3461..3508
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3527..3901
                     /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    4031..4152
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4068..4087)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4122..4141
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4201..4217)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4201..4217)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4214..4236)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4225..4241
                     /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(4249..4255,4256..4273,4274..4279))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4294..4315
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4432..4449)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4603..5191)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4683..4702)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5362..6153,6154..6222))
                     /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     5985..6004
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6223..6327)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
       61 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
      121 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
      181 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
      241 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
      301 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      361 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      421 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      481 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      541 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      601 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      661 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      721 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      781 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag
      841 agaacccact gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc
      901 tggctagcgt ttaaacttaa gcttggtacc gagctcggat ccactagtcc agtgtggtgg
      961 aattcgccac catggaccca gagacctgtc cgtgtccaac tggaggttct tgtacgtgta
     1021 gtgataagtg taaatgcaag ggatgtaaat gtactaactg taagaaatca tgttgtagtt
     1081 gttgccctgc cggatgtgag aaatgtgcga aagactgtgt ttgtaagggg gaagagggtg
     1141 ccaaagctga ggcggaaaaa tgttcctgtt gtcaaggtgg aggttctggc ggtggatcgg
     1201 gaggttcagc ggatcctgaa acatgtcctt gtcctactgg tggaagttgt acttgttctg
     1261 ataaatgtaa gtgcaaaggc tgcaaatgta ccaattgtaa aaaatcttgt tgttcgtgtt
     1321 gtcccgctgg ttgtgaaaag tgtgctaaag attgtgtatg taaaggtgaa gaaggagcaa
     1381 aagcagaagc cgagaagtgt tcatgctgtc agggcagcgg cccgggccct gacttcctgg
     1441 gccacgccga gaaccccctg agagaagagg aatgggccag actgaacgag acagtgatcc
     1501 aggtggccag gcggagcctc gtgggcagaa gaatcctgga catctacggc cctctgggcg
     1561 ctggcgtgca gacagtgcct tacgatgagt tccagggcgt gtcccctggc gccgtggata
     1621 ttgtgggcga gcaggaaacc gctatggtgt tcaccgacgc cagaaagttc aagaccatcc
     1681 ctattatcta caaggacttc ctgctgcact ggcgggacat cgaggccgcc agaacccaca
     1741 acatgcccct ggatgtgtct gccgctgctg gcgctgctgc tctgtgtgcc cagcaggaag
     1801 atgagctgat cttttacggc gacgccaggc tgggctacga gggcctgatg acagctaacg
     1861 gcagactgac cgtgcccctg ggcgattgga catctcctgg cggcggattc caggccatcg
     1921 tggaagccac cagaaagctg aacgagcagg gccacttcgg cccctacgct gtggtgctga
     1981 gccccagact gtacagccag ctgcacagaa tctacgaaaa gaccggcgtg ctggaaatcg
     2041 agacaatcag acagctggcc tccgacggcg tgtaccagag caacagactg agaggcgaga
     2101 gcggcgtggt ggtgtccacc ggcagagaaa acatggacct ggccgtgtct atggacatgg
     2161 tggccgctta cctgggcgcc tccagaatga accacccctt cagagtgctg gaagccctgc
     2221 tgctgagaat caagcacccc gacgccatct gcaccctgga aggcgcaggc gccacagaga
     2281 gaagagggtc aggagattac aaagatgacg atgataaata agcggccgct cgagtctaga
     2341 gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     2401 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     2461 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     2521 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     2581 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     2641 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2701 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2761 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     2821 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     2881 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     2941 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     3001 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     3061 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     3121 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     3181 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3241 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     3301 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     3361 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     3421 cgggagcttg tatatccatt ttcggatctg atcagcacgt gttgacaatt aatcatcggc
     3481 atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccatgg ccaagttgac
     3541 cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga
     3601 ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga
     3661 cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg
     3721 ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa
     3781 cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga
     3841 gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg
     3901 acacgtgcta cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     3961 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     4021 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     4081 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     4141 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     4201 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4261 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4321 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4381 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4441 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4501 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4561 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4621 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4681 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4741 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4801 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4861 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4921 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4981 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5041 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5101 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5161 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5221 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5281 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5341 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5401 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5461 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5521 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5581 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5641 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5701 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5761 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5821 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5881 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5941 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6001 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6061 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6121 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6181 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6241 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6301 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
//
