LOCUS       sequence-403816-        7944 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To generate LifeAct-mRuby expressing stable cell lines with piggybac
            transposase co-expression and blastocidin selections..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7944)
  AUTHORS   Liang X, Weberling A, Hii CY, Zernicka-Goetz M, Buckley CE
  TITLE     E-cadherin mediates apical membrane initiation site localisation 
            during de novo polarisation of epithelial cavities.
  JOURNAL   EMBO J. 2022 Aug 22:e111021. doi: 10.15252/embj.2022111021.
  PUBMED    35993232
REFERENCE   2  (bases 1 to 7944)
  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: "EMBO J. 
            2022 Aug 22:e111021. doi: 10.15252/embj.2022111021."
COMMENT     Sequence Label: PB-Blast-Dest-LifeAct-mRuby
FEATURES             Location/Qualifiers
     source          1..7944
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     434..453
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(672..776)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(802..1257)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(939..960)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1151..1170
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1384..1406
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1398..1415
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1399..1415
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1422..1441
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1422..1440
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   1459..1493
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    1768..1801
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        1811..2190
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2192..2468
                     /label=chicken beta-actin promoter
     intron          2469..3485
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3409..3431
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3493..3512
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    3549..3573
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3591..3641
                     /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             3663..4343
                     /codon_start=1
                     /product="monomeric variant of the red fluorescent protein 
                     eqFP611 (Kredel et al., 2009)"
                     /label=mRuby
                     /translation="MNSLIKENMRMKVVLEGSVNGHQFKCTGEGEGNPYMGTQTMRIKV
                     IEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGVITV
                     MQDTSLEDGCLVYHAQVRGVNFPSNGAVMQKKTKGWEPNTEMMYPADGGLRGYTHMALK
                     VDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVAKFA
                     GLGGG"
     protein_bind    complement(4348..4372)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    join(4524..5098,5099..5101,5102..5110)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4691..4708)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4918..4937
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     complement(5597..5616)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5662..5717
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5716..5735)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6078..6094)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6102..6118
                     /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(6126..6132,6133..6150,6151..6156))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6171..6192
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    6265..6298
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     repeat_region   6469..6531
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(6555..6573)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6594..6610)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6618..6634
                     /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(6642..6648,6649..6666,6667..6672))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6687..6708
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6825..6842)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6996..7584)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7076..7095)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7755..7944,1..602,603..671))
                     /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"
ORIGIN
        1 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
       61 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      121 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      181 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      241 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      301 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      361 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      421 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      481 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      541 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      601 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
      661 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
      721 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
      781 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
      841 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
      901 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
      961 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     1021 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     1081 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     1141 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     1201 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
     1261 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     1321 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     1381 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac tcactatagg
     1441 gcgaattggg taccttaacc ctagaaagat agtctgcgta aaattgacgc atgcattctt
     1501 gaaatattgc tctctctttc taaatagcgc gaatccgtcg ctgtgcattt aggacatctc
     1561 agtcgccgct tggagctccc gtgaggcgtg cttgtcaatg cggtaagtgt cactgatttt
     1621 gaactataac gaccgcgtga gtcaaaatga cgcatgatta tcttttacgt gacttttaag
     1681 atttaactca tacgataatt atattgttat ttcatgttct acttacgtga taacttatta
     1741 tatatatatt ttcttgttat agatatcata acttcgtata atgtatgcta tacgaagtta
     1801 tgcggccctc gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
     1861 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     1921 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     1981 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     2041 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     2101 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     2161 atctacgtat tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact
     2221 ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt
     2281 tgtgcagcga tgggggsggg gggggggggg ggsgcgcgcc rggsggggsg gggsggggsg
     2341 rggggsgggg cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc
     2401 gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc
     2461 ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc
     2521 gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc
     2581 cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc
     2641 tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg cggggggagc ggctcggggg
     2701 gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct
     2761 gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg
     2821 cggccggggg cggtgccccg cggtgcgggg ggggctgcga ggggaacaaa ggctgcgtgc
     2881 ggggtgtgtg cgtggggggg tgagcagggg gtgtgggcgc gtcggtcggg ctgcaacccc
     2941 ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg gggctccgta
     3001 cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg
     3061 gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc ggcccccgga
     3121 gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg taatcgtgcg
     3181 agagggcgca gggactttct ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc
     3241 gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg
     3301 gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc cagcctcggg
     3361 gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg ttcggcttct
     3421 ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc ttctttttcc
     3481 tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagaattcc
     3541 tcgaatcaac aagtttgtac aaaaaagcag gcttcgaagg agatagaacc atgggcgtgg
     3601 ccgacctgat caagaagttc gagagcatca gcaaggagga gccggatcca ccggtcgcca
     3661 ccatgaacag cctgatcaaa gaaaacatgc ggatgaaggt ggtgctggaa ggcagcgtga
     3721 acggccacca gttcaagtgc accggcgagg gcgagggcaa cccctacatg ggcacccaga
     3781 ccatgcggat caaagtgatc gagggcggac ctctgccctt cgccttcgac atcctggcca
     3841 catccttcat gtacggcagc cggaccttca tcaagtaccc caagggcatc cccgatttct
     3901 tcaagcagag cttccccgag ggcttcacct gggagagagt gaccagatac gaggacggcg
     3961 gcgtgatcac cgtgatgcag gacaccagcc tggaagatgg ctgcctggtg taccatgccc
     4021 aggtcagggg cgtgaatttt cccagcaacg gcgccgtgat gcagaagaaa accaagggct
     4081 gggagcccaa caccgagatg atgtaccccg ctgacggcgg actgagaggc tacacccaca
     4141 tggccctgaa ggtggacggc ggagggcacc tgagctgcag cttcgtgacc acctaccgat
     4201 ccaagaaaac cgtgggcaac atcaagatgc ccggcatcca cgccgtggac caccggctgg
     4261 aaaggctgga agagtccgac aacgagatgt tcgtggtgca gcgggagcac gccgtggcca
     4321 agttcgccgg cctgggcgga gggtaacacc cagctttctt gtacaaagtg gttgattcga
     4381 ggccgctcga taagcttgat atcgaattca ctcctcaggt gcaggctgcc tatcagaagg
     4441 tggtggctgg tgtggccaac tggctcacaa ataccactga gatcgacggt atcgataagc
     4501 ttgatatcga attccgcccc ccccccctct ccctcccccc cccctaacgt tactggccga
     4561 agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg
     4621 tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg
     4681 ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt
     4741 cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac
     4801 cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca
     4861 aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg
     4921 ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg
     4981 ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa
     5041 cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatat
     5101 ggccacaacc atgcctttgt ctcaagaaga atccaccctc attgaaagag caacggctac
     5161 aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc tctctagcga
     5221 cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt gtgcagaact
     5281 cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat
     5341 cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgccgacagg tgcttctcga
     5401 tctgcatcct gggatcaaag ccatagtgaa ggacagtgat ggacagccga cggcagttgg
     5461 gattcgtgaa ttgctgccct ctggttatgt gtgggagggt taactagata agtaatgatc
     5521 ataatcagcc atatcacatc tgtagaggtt ttacttgctt tgtccgacgc ggcccgacgg
     5581 gtccgagggg ggtcgatctt tttccctctg ccaaaaatta tggggacatc atgaagcccc
     5641 ttgagcatct gacttctggc taataaagga aatttatttt cattgcaata gtgtgttgga
     5701 attttttgtg tctctcactc ggaaggacat atgggagggc aaatcattta aaacatcaga
     5761 atgagtattt ggtttagagt ttggcaacat atgcccatat gctggctgcc atgaacaaag
     5821 gttggctata aagaggtcat cagtatatga aacagccccc tgctgtccat tccttattcc
     5881 atagaaaagc cttgacttga ggttagattt tttttatatt ttgttttgtg ttattttttt
     5941 ctttaacatc cctaaaattt tccttacatg ttttactagc cagatttttc ctcctctcct
     6001 gactactccc agtcatagct gtccctcttc tcttatggag atccctcgac ctgcagccca
     6061 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     6121 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     6181 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     6241 cagcggatcc gcggccgcgt cgagataact tcgtataatg tatgctatac gaagttatgt
     6301 tttgttactt tatagaagaa attttgagtt tttgtttttt tttaataaat aaataaacat
     6361 aaataaattg tttgttgaat ttattattag tatgtaagtg taaatataat aaaacttaat
     6421 atctattcaa attaataaat aaacctcgat atacagaccg ataaaacaca tgcgtcaatt
     6481 ttacgcatga ttatctttaa cgtacgtcac aatatgatta tctttctagg gttaaagagc
     6541 tccagctttt gttcccttta gtgagggtta atttcgagct tggcgtaatc atggtcatag
     6601 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     6661 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     6721 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     6781 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     6841 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     6901 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     6961 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     7021 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     7081 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     7141 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     7201 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     7261 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     7321 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     7381 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     7441 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     7501 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     7561 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     7621 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     7681 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     7741 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     7801 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     7861 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     7921 ttatcagcaa taaaccagcc agcc
//
