LOCUS       sequence-382845-        5860 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Labeling microtubules in living mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5860)
  AUTHORS   Burute M, Jansen KI, Mihajlovic M, Vermonden T, Kapitein LC
  TITLE     Local changes in microtubule network mobility instruct neuronal 
            polarization and axon specification.
  JOURNAL   Sci Adv. 2022 Nov 4;8(44):eabo2343. doi: 10.1126/sciadv.abo2343. 
            Epub 2022 Nov 4.
  PUBMED    36332030
REFERENCE   2  (bases 1 to 5860)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Adv."; 
            date: "2022-11-4"; volume: "8(44):eabo2343. doi"; pages: " 
            10.1126/sciadv.abo2343. Epub 2022 Nov 4"
COMMENT     Sequence Label: LifeAct-mCherry
FEATURES             Location/Qualifiers
     source          1..5860
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     146..165
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     399..416
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(568..573,574..591,592..598)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    606..622
                     /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)."
     primer_bind     611..633
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     630..646
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     630..646
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    673..807
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(697..716)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     751..770
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(929..1633)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYGAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1204..1223)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1473..1491
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(1643..1693)
                     /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"
     regulatory      1690..1699
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          1727..2553
                     /label=CMV intron A
                     /note="human cytomegalovirus intron A (Chapman et al., 
                     1991)"
     primer_bind     complement(2668..2692)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(2689..2892)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2718..2738)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2893..3272
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(3838..3855)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3838..3854)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3847..3869)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4101..4120)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4220..4242
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4280..4298)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             join(4486..4554,4555..5346)
                     /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     complement(4704..4723)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5517..5860,1..245)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
       61 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      121 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      181 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      241 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      301 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      361 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      421 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
      481 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtag
      541 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
      601 tggaattgtg agcggataac aatttcacac aggaaacagc tatgacatga ttacgcgagc
      661 tcggtacgga tcgatccaga catgataaga tacattgatg agtttggaca aaccacaact
      721 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
      781 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
      841 gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg
      901 gctgattatg atcaatgaat tatctagact tgtacagctc gtccatgccg ccggtggagt
      961 ggcggccctc ggcgcgttcg tactgttcca cgatggtgta gtcctcgttg tgggaggtga
     1021 tgtccaactt gatgttgacg ttgtaggcgc cgggcagctg cacgggcttc ttggccttgt
     1081 aggtggtctt gacctcagcg ccgtagtggc cgccgtcctt cagcttcagc ctctgcttga
     1141 tctcgccctt cagggcgccg tcctcggggt acatccgctc ggaggaggcc tcccagccca
     1201 tggtcttctt ctgcattacg gggccgtcgg aggggaagtt ggtgccgcgc agcttcacct
     1261 tgtagatgaa ctcgccgtcc tgcagggagg agtcctgggt cacggtcacc acgccgccgt
     1321 cctcgaagtt catcacgcgc tcccacttga agccctcggg gaaggacagc ttcaagtagt
     1381 cggggatgtc ggcggggtgc ttcacgtagg ccttggagcc gtacatgaac tgaggggaca
     1441 ggatgtccca ggcgaagggc agggggccac ccttggtcac cttcagcttg gcggtctggg
     1501 tgccctcgta ggggcggccc tcgccctcgc cctcgatctc gaactcgtgg ccgttcacgg
     1561 agccctccat gtgcaccttg aagcgcatga actccttgat gatggccatg ttatcctcct
     1621 cgcccttgct cactggatcc ccttcttcct ttgagatgct ttcgaatttc ttgatcaaat
     1681 ctgcgacacc catggtggct agcaagcttc gtgtcaagga cggtgactgc agaaaagacc
     1741 catggaaagg aacagtctgt tagtctgtca gctattatgt ctggtggcgc gcgcggcagc
     1801 aacgagtact gctcagacta cactgccctc caccgttaac agcaccgcaa cgggagttac
     1861 ctctgactct tatcagaaca caacaactca gctgcctgca tcttcttctg ccgctgcctt
     1921 aagtcttcca aatgcgtcag cggtgcaagc ccgctccccg agctcatttt cagacacata
     1981 ccctaccgcc acggccttgt gcggcacact ggtggtggtg ggcatcgtgc tgtgcctaag
     2041 tctggcctcc actgttagga gcaaggagct gccgagcgac catgagtcgc tggaggcatg
     2101 ggagcagggc tcggatgtag aagctccgcc gctaccggag aagagcccat gtccggaaca
     2161 cgtacccgag attcgcgtgg agatcccacg ttatgtttaa taaaaactgc gggcactggg
     2221 gacggtggtg ttgtatatgt gaatttgtaa ataataaatg agaccccatc ctgtaaaaat
     2281 acagagtccg tgtcagtctc tgaaggacag tgtattggca tatagccaat aaagagagtt
     2341 gtggcaaaga gccatgttat ggattagtaa tggaaagtat cgtcaccaat aggggagtgg
     2401 tcaataatgg tcaataaccc acacctatag gctaagctat accatcacct ataacatgag
     2461 gaagcggggg tgtatagacc ccaagccaaa aacagtatag catgcataag aagccaaggg
     2521 ggtgggccta tagactctat aggcggtact tacgtcactc ttggcacggg gaatccgcgt
     2581 tccaatgcac cgttcccggc cgcggaggct ggatcggtcc cggtgtcttc tatggaggtc
     2641 aaaacagcgt ggatggcgtc tccaggcgat ctgacggttc actaaacgag ctctgcttat
     2701 atagacctcc caccgtacac gcctaccgcc catttgcgtc aatggggcgg agttgttacg
     2761 acattttgga aagtcccgtt gattttggtg ccaaaacaaa ctcccattga cgtcaatggg
     2821 gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca cgcccattga tgtactgcca
     2881 aaaccgcatc accatggtaa tagcgatgac taatacgtag atgtactgcc aagtaggaaa
     2941 gtcccataag gtcatgtact gggcataatg ccaggcgggc catttaccgt cattgacgtc
     3001 aatagggggc gtacttggca tatgatacac ttgatgtact gccaagtggg cagtttaccg
     3061 taaatactcc acccattgac gtcaatggaa agtccctatt ggcgttacta tgggaacata
     3121 cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt cagccaggcg ggccatttac
     3181 cgtaagttat gtaacgcgga actccatata tgggctatga actaatgacc ccgtaattga
     3241 ttactattaa taactagtca ataatcaatg tcaacatggc ggtaatgttg gacatgagcc
     3301 aatataaatg tacatattat gatatggata caacgtatgc aatggccaat agccaatatt
     3361 gatttatgct atataaccaa tgaataatat ggctaatggc caatattgat tcaatgtata
     3421 gatcgatatg cattggccat gtgccagctt gatgtcgcct ctatcggcga tatagcctca
     3481 tatcgtctgt cacctatatc gaaactgcga tatttgcgac acacagaatc gcccaagtca
     3541 ccaaagtcgt ctatcgccat cccccgtaaa cgatataagc gctatcgcca gatatcgcgt
     3601 atgcccaaaa atcacttttg gaaaaatggc gatatcagtt acacagaaac tcacatcggc
     3661 gacattttca atatgccata ttttcaaata tcgatttttc caatatcgcc atctctatcg
     3721 gcgataaaca ccactatcgc gcgacatgaa tttagtcggc gacagaaatc tcaaaacgcg
     3781 tatttcggac aaacacacat tttattattc actgcagtca ccgtccttga cacgggcact
     3841 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     3901 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     3961 ttcccaacag ttgcgcagcc tgaatggcga atggcgccct gactaatttt ttttatttat
     4021 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggcgacgtc
     4081 ttcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     4141 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     4201 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     4261 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     4321 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     4381 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     4441 atgcttcaat aataactcga ggccgcttat tgaaaaagga agagtatgag tattcaacat
     4501 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     4561 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     4621 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     4681 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     4741 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     4801 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     4861 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     4921 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     4981 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     5041 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     5101 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     5161 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     5221 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     5281 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     5341 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     5401 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     5461 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     5521 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5581 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5641 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     5701 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5761 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5821 cagcggtcgg gctgaacggg gggttcgtgc acacagccca
//
