LOCUS       sequence-350942-        7790 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Lifeact-miRFP703 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7790)
  AUTHORS   Yamamoto K, Miura H, Ishida M, Mii Y, Kinoshita N, Takada S, Ueno N,
            Sawai S, Kondo Y, Aoki K
  TITLE     Optogenetic relaxation of actomyosin contractility uncovers 
            mechanistic roles of cortical tension during cytokinesis.
  JOURNAL   Nat Commun. 2021 Dec 8;12(1):7145. doi: 10.1038/s41467-021-27458-3.
  PUBMED    34880255
REFERENCE   2  (bases 1 to 7790)
  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 
            Commun."; date: "2021-12-8"; volume: "12(1):7145. doi"; pages: " 
            10.1038/s41467-021-27458-3"
COMMENT     Sequence Label: pPBbsr2-Lifeact-miRFP703
FEATURES             Location/Qualifiers
     source          1..7790
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   674..708
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        1011..1390
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1668..2684
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2608..2630
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2692..2711
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2754..2804
                     /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"
     CDS             2814..3758
                     /codon_start=1
                     /product="bright monomeric near-infrared fluorescent 
                     protein engineered from a bacterial phytochrome, with an 
                     emission maximum of 703 nm (Shcherbakova et al., 2016)"
                     /label=miRFP703
                     /label=ORF frame 1
                     /translation="MVAGHASGSPAFGTASHSNCEHEEIHLAGSIQPHGALLVVSEHDH
                     RVIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTE
                     YCGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTG
                     YDRVMVYRFDEQGHGLVFSECHVPGLESYFGNRYPSSLVPQMARQLYVRQRVRVLVDVT
                     YQPVPLEPRLSPLTGRDLDMSGCFLRSMSPIHLQFLKDMGVRATLAVSLVVGGKLWGLV
                     VCHHYLPRFIRFELRAICKRLAERIATRITALES"
     misc_feature    3854..4403
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3998..4015)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4225..4244
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     complement(4874..4891)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4880..5104
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5151..5167)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5175..5191
                     /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(5199..5205,5206..5223,5224..5229))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5244..5265
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   5499..5561
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5602..5622)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5602..5620)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5641..5657)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5665..5681
                     /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(5689..5695,5696..5713,5714..5719))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5734..5755
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5872..5889)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6043..6631)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6123..6142)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6802..7593,7594..7662))
                     /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     7425..7444
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7663..7767)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggggcgcgc
      661 cattctagat taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat
      721 attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg
      781 ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact
      841 ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta
      901 actcatacga taattatatt gttatttcat gttctactta cgtgataact tattatatat
      961 atattttctt gttatagata tcaactagaa tgctagcatg ggcccatctc gacattgatt
     1021 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     1081 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     1141 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     1201 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     1261 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     1321 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     1381 tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc
     1441 cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg
     1501 gggggggggg gsgcgcgccr ggsggggsgg ggsggggsgr ggggsggggs ggggsgrggc
     1561 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga
     1621 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc
     1681 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac
     1741 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
     1801 agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc
     1861 tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg
     1921 tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg
     1981 cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc
     2041 ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt
     2101 gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc cctccccgag
     2161 ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg
     2221 ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg
     2281 ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg
     2341 cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt
     2401 tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc
     2461 gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt
     2521 cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct
     2581 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta
     2641 gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc
     2701 tggttattgt gctgtctcat cattttggca aagaattggg atccgaattc gccatgggtg
     2761 tagcagatct catcaagaag ttcgaatcca tctccaagga ggagggcggc cgcatggtag
     2821 caggtcatgc ctctggcagc cccgcattcg ggaccgcctc tcattcgaat tgcgaacatg
     2881 aagagatcca cctcgccggc tcgatccagc cgcatggcgc gcttctggtc gtcagcgaac
     2941 atgatcatcg cgtcatccag gccagcgcca acgccgcgga atttctgaat ctcggaagcg
     3001 tactcggcgt tccgctcgcc gagatcgacg gcgatctgtt gatcaagatc ctgccgcatc
     3061 tcgatcccac cgccgaaggc atgccggtcg cggtgcgctg ccggatcggc aatccctcta
     3121 cggagtactg cggtctgatg catcggcctc cggaaggcgg gctgatcatc gaactcgaac
     3181 gtgccggccc gtcgatcgat ctgtcaggca cgctggcgcc ggcgctggag cggatccgca
     3241 cggcgggttc actgcgcgcg ctgtgcgatg acaccgtgct gctgtttcag cagtgcaccg
     3301 gctacgaccg ggtgatggtg tatcgtttcg atgagcaagg ccacggcctg gtattctccg
     3361 agtgccatgt gcctgggctc gaatcctatt tcggcaaccg ctatccgtcg tcgctggtcc
     3421 cgcagatggc gcggcagctg tacgtgcggc agcgcgtccg cgtgctggtc gacgtcacct
     3481 atcagccggt gccgctggag ccgcggctgt cgccgctgac cgggcgcgat ctcgacatgt
     3541 cgggctgctt cctgcgctcg atgtcgccga tccatctgca gttcctgaag gacatgggcg
     3601 tgcgcgccac cctggcggtg tcgctggtgg tcggcggcaa gctgtggggc ctggttgtct
     3661 gtcaccatta tctgccgcgc ttcatccgtt tcgagctgcg ggcgatctgc aaacggctcg
     3721 ccgaaaggat cgcgacgcgg atcaccgcgc ttgagagctc tagataagtc gacgggccgc
     3781 ggtaacaatt gttaactaac ttaagctagc aacggtttcc ctctagcggg atcaattccg
     3841 cccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc
     3901 tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc
     3961 cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt
     4021 ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct
     4081 gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa
     4141 aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt
     4201 tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag
     4261 gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt
     4321 acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt
     4381 ttcctttgaa aaacacgata ataccatggt catgaaaaca tttaacattt ctcaacaaga
     4441 tctagaatta gtagaagtag cgacagagaa gattacaatg ctttatgagg ataataaaca
     4501 tcatgtggga gcggcaattc gtacgaaaac aggagaaatc atttcggcag tacatattga
     4561 agcgtatata ggacgagtaa ctgtttgtgc agaagccatt gcgattggta gtgcagtttc
     4621 gaatggacaa aaggattttg acacgattgt agctgttaga cacccttatt ctgacgaagt
     4681 agatagaagt attcgagtgg taagtccttg tggtatgtgt agggagttga tttcagacta
     4741 tgcaccagat tgttttgtgt taatagaaat gaatggcaag ttagtcaaaa ctacgattga
     4801 agaactcatt ccactcaaat atacccgaaa ttaaaagtcg agatgcatcg atgatctaga
     4861 gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     4921 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     4981 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     5041 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     5101 atggcttctg aggcggaaag aacctgcagc ccaagcttgg cgtaatcatg gtcatagctg
     5161 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     5221 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     5281 ctgcccgctt tccagtcggg aaacctgtcg tgccagcgga tcttaaaagt tttgttactt
     5341 tatagaagaa attttgagtt tttgtttttt tttaataaat aaataaacat aaataaattg
     5401 tttgttgaat ttattattag tatgtaagtg taaatataat aaaacttaat atctattcaa
     5461 attaataaat aaacctcgat atacagaccg ataaaacaca tgcgtcaatt ttacgcatga
     5521 ttatctttaa cgtacgtcac aatatgatta tctttctagg gttaatctag tatacgcgtt
     5581 taattaatcc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg
     5641 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     5701 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     5761 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5821 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5881 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5941 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     6001 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     6061 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     6121 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     6181 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     6241 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     6301 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6361 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6421 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6481 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6541 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6601 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6661 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6721 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6781 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6841 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6901 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6961 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     7021 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     7081 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     7141 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     7201 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     7261 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     7321 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     7381 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7441 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7501 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7561 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7621 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7681 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7741 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
//
