LOCUS       sequence-442823-        5463 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses LifeAct-mScarlet-MTED for targeting fluorescent, MT 
            depolymerizing peptide MTED to f-actin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5463)
  AUTHORS   Holland ED, Miller HL, Millette MM, Taylor RJ, Drucker GL, Dent EW
  TITLE     A methodology for specific disruption of microtubule polymerization 
            into dendritic spines.
  JOURNAL   Mol Biol Cell. 2024 Jun 1;35(6):mr3. doi: 10.1091/mbc.E24-02-0093. 
            Epub 2024 Apr 17.
  PUBMED    38630519
REFERENCE   2  (bases 1 to 5463)
  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: "Mol Biol 
            Cell."; date: "2024-06-1"; volume: "35(6):mr3. doi"; pages: " 
            10.1091/mbc.E24-02-0093. Epub 2024 Apr 17"
COMMENT     Sequence Label: LifeAct-MTED
FEATURES             Location/Qualifiers
     source          1..5463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    117..251
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(154..173)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     208..227
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(319..336)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(490..1078)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(570..589)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1249..2040,2041..2109))
                     /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     1872..1891
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2110..2214)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2270..2584
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    2278..2296
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2314..2332
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2349..2367
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2385..2403
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2421..2439
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2456..2474
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2492..2510
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2557..2581
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2610..2619
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2616..2666
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     promoter        3476..3986
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3905..3923
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             join(3996..4613,4614..4619,4620..4655,4656..4658,4659..4694,
                     4695..4697,4698..4733,4734..4742)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     polyA_signal    4790..4845
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4844..4863)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5206..5222)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5206..5222)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5219..5241)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5230..5246
                     /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(5254..5260,5261..5278,5279..5284))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5299..5320
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5379..5463,1..111)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      join(5425..5463,1..97)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
       61 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctaact
      121 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
      181 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
      241 atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
      301 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
      361 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
      421 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
      481 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
      541 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
      601 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
      661 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
      721 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
      781 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
      841 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
      901 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
      961 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     1021 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     1081 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     1141 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     1201 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     1261 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     1321 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     1381 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     1441 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     1501 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     1561 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     1621 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     1681 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     1741 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     1801 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     1861 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     1921 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     1981 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     2041 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     2101 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     2161 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     2221 tccccgaaaa gtgccacctg gtcgtcgact ggccgcgaat cgatatgtcg agtttactcc
     2281 ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgatgtc
     2341 gagtttactc cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag
     2401 agaacgtatg tcgagtttac tccctatcag tgatagagaa cgtatgtcga gtttatccct
     2461 atcagtgata gagaacgtat gtcgagttta ctccctatca gtgatagaga acgtatgtcg
     2521 aggtaggcgt gtacggtggg aggcctatat aagcagagct cgtttagtga accgtcagat
     2581 cgcctggaga aaccggttat actgcggccg ccaccatggg cgtggcggat ctgattaaga
     2641 agttcgaaag cattagcaaa gaagaaggct ctggttccgg cactagtgtg agtaaaggag
     2701 aggccgttat aaaagagttc atgcggttca aggtacacat ggagggatca atgaacggac
     2761 acgagttcga aatcgaaggg gaaggagagg gcaggcccta tgagggtact cagaccgcca
     2821 aactcaaagt gacaaaggga ggccctttgc ctttctcttg ggacatcttg tcaccacaat
     2881 ttatgtatgg ctcccgagca tttataaaac acccagcaga catacctgat tactacaagc
     2941 agtctttccc agaaggtttt aaatgggaaa gggtcatgaa ctttgaggat ggtggtgctg
     3001 tgacggtgac gcaggatact tctttggagg acggaacact catctacaag gtaaagctca
     3061 ggggcacgaa cttcccgccg gacggaccag tcatgcagaa aaagactatg ggatgggagg
     3121 cttccaccga acggttgtat cccgaggacg gtgtcttgaa gggagatata aaaatggctc
     3181 ttcggctgaa agatggaggc agatacttgg cagattttaa gaccacgtat aaggcaaaaa
     3241 aaccagtgca aatgccggga gcatataacg ttgaccgcaa actggacatc acctcacata
     3301 acgaggatta tacagtggta gaacagtacg aaagaagcga agggaggcac agcaccgggg
     3361 gtatggatga gctgtataag ggcagcggca gcggcagcgc gccgcgcttt gaagcgtata
     3421 tgatgaccgg cgatctgatt ctgaacctga gccgcacctg aacgcgtcat gctccggggt
     3481 tggggttgcg ccttttccaa ggcagccctg ggtttgcgca gggacgcggc tgctctgggc
     3541 gtggttccgg gaaacgcagc ggcgccgacc ctgggtctcg cacattcttc acgtccgttc
     3601 gcagcgtcac ccggatcttc gccgctaccc ttgtgggccc cccggcgacg cttcctgctc
     3661 cgcccctaag tcgggaaggt tccttgcggt tcgcggcgtg ccggacgtga caaacggaag
     3721 ccgcacgtct cactagtacc ctcgcagacg gacagcgcca gggagcaatg gcagcgcgcc
     3781 gaccgcgatg ggctgtggcc aatagcggct gctcagcagg gcgcgccgag agcagcggcc
     3841 gggaaggggc ggtgcgggag gcggggtgtg gggcggtagt gtgggccctg ttcctgcccg
     3901 cgcggtgttc cgcattctgc aagcctccgg agcgcacgtc ggcagtcggc tccctcgttg
     3961 accgaatcac cgacctctct ccccagcaat tcaccatgtc tagactggac aagagcaaag
     4021 tcataaacgg agctctggaa ttactcaatg gtgtcggtat cgaaggcctg acgacaagga
     4081 aactcgctca aaagctggga gttgagcagc ctaccctgta ctggcacgtg aagaacaagc
     4141 gggccctgct cgatgccctg ccaatcgaga tgctggacag gcatcatacc cacttctgcc
     4201 ccctggaagg cgagtcatgg caagactttc tgcggaacaa cgccaagtca taccgctgtg
     4261 ctctcctctc acatcgcgac ggggctaaag tgcatctcgg cacccgccca acagagaaac
     4321 agtacgaaac cctggaaaat cagctcgcgt tcctgtgtca gcaaggcttc tccctggaga
     4381 acgcactgta cgctctgtcc gccgtgggcc actttacact gggctgcgta ttggaggaac
     4441 aggagcatca agtagcaaaa gaggaaagag agacacctac caccgattct atgcccccac
     4501 ttctgagaca agcaattgag ctgttcgacc ggcagggagc cgaacctgcc ttccttttcg
     4561 gcctggaact aatcatatgt ggcctggaga aacagctaaa gtgcgaaagc ggcgggccga
     4621 ccgacgccct tgacgatttt gacttagaca tgctcccagc cgatgccctt gacgactttg
     4681 accttgatat gctgcctgct gacgctcttg acgattttga ccttgacatg ctccccgggt
     4741 aactaagtaa ggatcgatcc aaggtacctt gagcatctga cttctggcta ataaaggaaa
     4801 tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat
     4861 gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat
     4921 gcccatatgc tggctgccat gaacaaaggt tggctataaa gaggtcatca gtatatgaaa
     4981 cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt
     5041 tttatatttt gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt
     5101 ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc
     5161 ttatggagat ccctcgacct gcagcccaag cttggcgtaa tcatggtcat agctgtttcc
     5221 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     5281 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5341 cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc
     5401 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     5461 ccg
//
