LOCUS       sequence-311954-        8347 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Shield-1 inducible transient mammalian expression of 
            Dam-tdTomato-LMNB1 for DamID, containing the V133A mutant of Dam and
            a fused tdTomato to monitor fusion protein localization and 
            expression levels..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8347)
  AUTHORS   Altemose N, Maslan A, Rios-Martinez C, Lai A, White JA, Streets A
  TITLE     muDamID: A Microfluidic Approach for Joint Imaging and Sequencing of
            Protein-DNA Interactions in Single Cells.
  JOURNAL   Cell Syst. 2020 Oct 21;11(4):354-366.e9. doi: 
            10.1016/j.cels.2020.08.015. Epub 2020 Sep 23.
  PUBMED    33099405
REFERENCE   2  (bases 1 to 8347)
  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"; doi: 
            "10.1016/j.cels.2020.08.015"; journalName: "Cell Syst"; date: 
            "2020-10-21- 21"; volume: "11"; issue: "4"; pages: "354-366.e9"
COMMENT     Sequence Label: DD-DamV133A-tdTomato-LMNB1
FEATURES             Location/Qualifiers
     source          1..8347
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(537..539,540..860)
                     /codon_start=1
                     /product="destabilization domain that can be stabilized by 
                     Shield1 in the ProteoTuner(TM) system"
                     /label=DD
                     /note="L106P mutant of FKBP12"
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKPE"
     CDS             1746..1787
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    1803..1827
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1833..1842
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1839..3266
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     polyA_signal    5075..5196
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5112..5131)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5166..5185
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5203..5658)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5340..5361)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5552..5571
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5685..5789
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5791..6148
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5791..5982
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5814..5834)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5999..6134
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6183..6977
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             6183..6977
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6237..6256)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6847..6866
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7165..7184)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7209..7256
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7585..8173
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8074..8093
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcatgg
      541 gagtgcaggt ggaaaccatc tccccaggag acgggcgcac cttccccaag cgcggccaga
      601 cctgtgtggt gcactacacc gggatgcttg aagatggaaa gaaagtcgat tcctcccggg
      661 acagaaacaa gccctttaag tttatgctag gcaagcagga ggtgatccga ggctgggaag
      721 aaggggttgc ccagatgagt gtgggtcaga gagccaaact gactatatct ccagattatg
      781 cctatggtgc cactgggcac ccaggcatca tcccaccaca tgccactctc gtcttcgatg
      841 tggagcttct aaaaccggaa gctagcgcta ccggactcag atcttcacac acagctagcg
      901 ccaccatgaa gaaaaatcgc gcttttttga agtgggcagg gggcaagtat cccctgcttg
      961 atgatattaa acggcatttg cccaagggcg aatgtctggt tgagcctttt gtaggtgccg
     1021 ggtcggtgtt tctcaacacc gacttttctc gttatatcct tgccgatatc aatagcgacc
     1081 tgatcagtct ctataacatt gtgaagatgc gtactgatga gtacgtacag gccgcacgcg
     1141 agctgtttgt tcccgaaaca aattgcgccg aggtttacta tcagttccgc gaagagttca
     1201 acaaaagcca ggatccgttc cgtcgggcgg tactgttttt atatttgaac cgctacggtt
     1261 acaacggcct gtgtcgttac aatctgcgcg gtgagtttaa cgcgccgttc ggccgctaca
     1321 aaaaacccta tttcccggaa gcagagttgt atcacttcgc tgaaaaagcg cagaatgcct
     1381 ttttctattg tgagtcttac gccgatagca tggcgcgcgc agatgatgca tccgtcgtct
     1441 attgcgatcc gccttatgca ccgctgtctg cgaccgccaa ctttacggcg tatcacacaa
     1501 acagttttac gcttgaacaa caagcgcatc tggcggagat cgccgaaggt ctggttgagc
     1561 gccatattcc agtgctgatc tccaatcacg atacgatgtt aacgcgtgag tggtatcagc
     1621 gcgcaaaatt gcatgtcgtc aaagttcgac gcagtataag cagcaacggc ggcacacgta
     1681 aaaaggtgga cgaactgctg gctttgtaca aaccaggagt cgtttcaccc gcgaaaaaaa
     1741 ccggtggtaa gcctatccct aaccctctcc tcggtctcga ttctacgatc gatttcgatc
     1801 aaacaagttt gtacaaaaaa gcaggctccg cggccgccat ggtgagcaag ggcgaggagg
     1861 tcatcaaaga gttcatgcgc ttcaaggtgc gcatggaggg ctccatgaac ggccacgagt
     1921 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
     1981 aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtccccc cagttcatgt
     2041 acggctccaa ggcgtacgtg aagcaccccg ccgacatccc cgattacaag aagctgtcct
     2101 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggt ctggtgaccg
     2161 tgacccagga ctcctccctg caggacggca cgctgatcta caaggtgaag atgcgcggca
     2221 ccaacttccc ccccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcca
     2281 ccgagcgcct gtacccccgc gacggcgtgc tgaagggcga gatccaccag gccctgaagc
     2341 tgaaggacgg cggccactac ctggtggagt tcaagaccat ctacatggcc aagaagcccg
     2401 tgcaactgcc cggctactac tacgtggaca ccaagctgga catcacctcc cacaacgagg
     2461 actacaccat cgtggaacag tacgagcgct ccgagggccg ccaccacctg ttcctggggc
     2521 atggcaccgg cagcaccggc agcggcagct ccggcaccgc ctcctccgag gacaacaaca
     2581 tggccgtcat caaagagttc atgcgcttca aggtgcgcat ggagggctcc atgaacggcc
     2641 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
     2701 agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tccccccagt
     2761 tcatgtacgg ctccaaggcg tacgtgaagc accccgccga catccccgat tacaagaagc
     2821 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggtctgg
     2881 tgaccgtgac ccaggactcc tccctgcagg acggcacgct gatctacaag gtgaagatgc
     2941 gcggcaccaa cttccccccc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     3001 cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagatc caccaggccc
     3061 tgaagctgaa ggacggcggc cactacctgg tggagttcaa gaccatctac atggccaaga
     3121 agcccgtgca actgcccggc tactactacg tggacaccaa gctggacatc acctcccaca
     3181 acgaggacta caccatcgtg gaacagtacg agcgctccga gggccgccac cacctgttcc
     3241 tgtacggcat ggacgagctg tacaagtccg gactcagatc tcgagctcaa gcttcgatgg
     3301 cgactgcgac ccccgtgccg ccgcggatgg gcagccgcgc tggcggcccc accacgccgc
     3361 tgagccccac gcgcctgtcg cggctccagg agaaggagga gctgcgcgag ctcaatgacc
     3421 ggctggcggt gtacatcgac aaggtgcgca gcctggagac ggagaacagc gcgctgcagc
     3481 tgcaggtgac ggagcgcgag gaggtgcgcg gccgtgagct caccggcctc aaggcgctct
     3541 acgagaccga gctggccgac gcgcgacgcg cgctcgacga cacggcccgc gagcgcgcca
     3601 agctgcagat cgagctgggc aagtgcaagg cggaacacga ccagctgctc ctcaactatg
     3661 ctaagaagga atctgatctt aatggcgccc agatcaagct tcgagaatat gaagcagcac
     3721 tcaattcgaa agatgcagct cttgctactg cacttggtga caaaaaaagt ttagagggag
     3781 atttggagga tctgaaggat cagattgccc agttggaagc ctccttagct gcagccaaaa
     3841 aacagttagc agatgaaact ttacttaaag tagatttgga gaatcgttgt cagagcctta
     3901 ctgaggactt ggagtttcgc aaaagcatgt atgaagagga gattaacgag accagaagga
     3961 agcatgaaac gcgcttggta gaggtggatt ctgggcgtca aattgagtat gagtacaagc
     4021 tggcgcaagc ccttcatgag atgagagagc aacatgatgc ccaagtgagg ctgtataagg
     4081 aggagctgga gcagacttac catgccaaac ttgagaatgc cagactgtca tcagagatga
     4141 atacttctac tgtcaacagt gccagggaag aactgatgga aagccgcatg agaattgaga
     4201 gcctttcatc ccagctttct aatctacaga aagagtctag agcatgtttg gaaaggattc
     4261 aagaattaga ggacttgctt gctaaagaaa aagacaactc tcgtcgcatg ctgacagaca
     4321 aagagagaga gatggcggaa ataagggatc aaatgcagca acagctgaat gactatgaac
     4381 agcttcttga tgtaaagtta gccctggaca tggaaatcag tgcttacagg aaactcttag
     4441 aaggcgaaga agagaggttg aagctgtctc caagcccttc ttcccgtgtg acagtatccc
     4501 gagcatcctc aagtcgtagt gtacgtacaa ctagaggaaa gcggaagagg gttgatgtgg
     4561 aagaatcaga ggcgagtagt agtgttagca tctctcattc cgcctcagcc actggaaatg
     4621 tttgcatcga agaaattgat gttgatggga aatttatccg cttgaagaac acttctgaac
     4681 aggatcaacc aatgggaggc tgggagatga tcagaaaaat tggagacaca tcagtcagtt
     4741 ataaatatac ctcaagatat gtgctgaagg caggccagac tgttacaatt tgggctgcaa
     4801 acgctggtgt cacagccagc cccccaactg acctcatctg gaagaaccag aactcgtggg
     4861 gcactggcga agatgtgaag gttatattga aaaattctca gggagaggag gttgctcaaa
     4921 gaagtacagt ctttaaaaca accatacctg aagaagagga ggaggaggaa gaagcagctg
     4981 gagtggttgt tgaggaagaa cttttccacc agcagggaac cccaagagca tccaatagaa
     5041 gctgtgcaat tatgtagtga caattgttgt tgttaacttg tttattgcag cttataatgg
     5101 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     5161 tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta agcgttaata
     5221 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     5281 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     5341 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     5401 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     5461 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     5521 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     5581 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     5641 cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5701 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5761 tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt
     5821 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     5881 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     5941 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     6001 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     6061 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     6121 cttttttgga ggcctaggct tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc
     6181 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     6241 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     6301 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     6361 tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     6421 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     6481 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     6541 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     6601 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     6661 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg
     6721 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     6781 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     6841 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     6901 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     6961 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     7021 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     7081 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     7141 cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg
     7201 cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt ttgttcataa
     7261 acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg
     7321 ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc
     7381 agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt actcatatat
     7441 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     7501 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     7561 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     7621 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     7681 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     7741 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     7801 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     7861 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     7921 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     7981 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     8041 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     8101 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     8161 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     8221 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     8281 catgcattag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
     8341 agttccg
//
