LOCUS       sequence-448463-        7682 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express mEGFP-fusion protein of FXR1 isoform a with shRNA5-resistant
            silent mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7682)
  AUTHORS   Chen X, Fansler MM, Janjos U, Ule J, Mayr C
  TITLE     The FXR1 network acts as signaling scaffold for actomyosin 
            remodeling.
  JOURNAL   Cell. 5 August 2024. doi: 10.1016/j.cell.2024.07.015.
  PUBMED    37961296
REFERENCE   2  (bases 1 to 7682)
  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: "Cell. 5 
            August 2024. doi: 10.1016/j.cell.2024.07.015."
COMMENT     Sequence Label: pcDNA3puro-mGFP-FXR1a-sh5Resistant
FEATURES             Location/Qualifiers
     source          1..7682
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(119..223)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(298..317)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        489..868
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        869..1072
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1023..1043
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1117..1136
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1117..1135
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1159..1161,1162..1164,1165..1875)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1204..1225)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1465..1484)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1812..1833
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3776..3793)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3782..4006
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4052..4480
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4139..4158)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4349..4370
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4489..4509)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4494..4823
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4674..4809
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4736..4755
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4836..5435
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4836..4855)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5332..5352
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5609..5730
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5646..5665)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5700..5719
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5779..5795)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5779..5795)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5792..5814)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5803..5819
                     /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(5827..5833,5834..5851,5852..5857))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5872..5893
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6010..6027)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6181..6769)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6261..6280)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6940..7682,1..49,50..118))
                     /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     7563..7582
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
       61 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
      121 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
      181 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
      241 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
      301 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
      361 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
      421 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
      481 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
      541 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      601 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      661 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      721 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      781 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      841 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
      901 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
      961 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
     1021 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct
     1081 aactagagaa cccactgctt actggcttat cgaaattaat acgactcact atagggagac
     1141 ccaagctggc tagcaaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     1201 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     1261 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     1321 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     1381 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     1441 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     1501 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1561 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     1621 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     1681 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1741 ctgctgcccg acaaccacta cctgagcacc cagtccaagc tgagcaaaga ccccaacgag
     1801 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     1861 gacgagctgt acaaggcgga gctgacggtg gaggttcgcg gctctaacgg ggctttctac
     1921 aagggattta tcaaagatgt tcatgaagac tcccttacag ttgtttttga aaataattgg
     1981 caaccagaac gccaggttcc atttaatgaa gttagattac caccaccacc tgatataaaa
     2041 aaagaaatta gtgaaggaga tgaagtagag gtatattcaa gagcaaatga ccaagagcca
     2101 tgtgggtggt ggttggctaa agttcggatg atgaaaggag aattttatgt cattgaatat
     2161 gctgcttgtg acgctactta caatgaaata gtcacatttg aacgacttcg gcctgtcaat
     2221 caaaataaaa ctgtcaaaaa aaataccttc tttaaatgca cagtggatgt tcctgaggat
     2281 ttgagagagg cgtgtgctaa tgaaaatgca cataaagatt ttaagaaagc agtaggagca
     2341 tgcagaattt tttaccatcc agaaacaaca cagctaatga tactgtctgc cagtgaagca
     2401 actgtgaaga gagtaaacat cttaagtgac atgcatttgc gaagtattcg tacgaagttg
     2461 atgcttatgt ccagaaatga agaggccact aagcatttag aatgcacaaa acaacttgca
     2521 gcagcttttc atgaggaatt tgttgtgaga gaagatttaa tgggcctggc aataggaaca
     2581 catggtagta acatccagca agctaggaag gttcctggag ttaccgccat tgagctagat
     2641 gaagatactg gaacattcag aatctacgga gagagtgctg atgctgtaaa aaaggctaga
     2701 ggtttcttgg aatttgtgga ggattttatt caggttccta ggaatctcgt tggaaaagta
     2761 attggaaaaa atggcaaagt tattcaagaa atagtggaca aatctggtgt ggttcgagtg
     2821 agaattgaag gggacaatga aaataaatta cccagagaag acggtatggt tccatttgta
     2881 tttgttggca ctaaagaaag cattggaaat gtgcaggttc ttctagagta tcatattgcc
     2941 tatctaaagg aagtagaaca gctaagaatg gaacgcctac agattgatga acagctgcga
     3001 cagattggtt ctaggtctta tagcggaaga ggcagaggtc gtcggggacc taattacacc
     3061 tccggttatg gtacaaattc tgagctgtct aacccctctg aaacggaatc tgagcgtaaa
     3121 gacgagctga gtgattggtc attggcagga gaagatgatc gagacagccg acatcagcgt
     3181 gacagcagga gacgcccagg aggaagaggc agaagtgttt cagggggtcg aggtcgtggt
     3241 ggaccacgtg gtggcaaatc ctccatcagt tctgtgctca aagatccaga cagcaatcca
     3301 tacagcttac ttgataatac agaatcagat cagactgcag acactgatgc atccgaaagc
     3361 caccattcca ctaaccgtcg taggcggtct cgtagacgaa ggactgatga agatgctgtt
     3421 ctgatggatg gaatgactga atctgataca gcttcagtta atgaaaatgg gctagtcaca
     3481 gttgcagatt atatttctag agctgagtct cagagcagac aaagaaacct cccaagggaa
     3541 actttggcta aaaacaagaa agaaatggca aaagatgtga ttgaagagca tggtccttca
     3601 gaaaaggcaa taaacggccc aactagtgct tctggcgatg acatttctaa gctacagcgt
     3661 actccaggag aagaaaagat taatacctta aaagaagaaa acactcaaga agcagcagtc
     3721 ctgaatggtg tttcataact cgagtctaga gggcccgttt aaacccgctg atcagcctcg
     3781 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     3841 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     3901 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     3961 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     4021 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     4081 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     4141 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     4201 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     4261 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     4321 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     4381 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     4441 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
     4501 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     4561 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     4621 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     4681 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     4741 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     4801 ttggaggcct aggcttttgc aaaaagctcc caaccatgac cgagtacaag cccacggtgc
     4861 gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg
     4921 actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc
     4981 tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg
     5041 acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg
     5101 ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga
     5161 tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg
     5221 gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg
     5281 aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc
     5341 ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc
     5401 gcacctggtg catgacccgc aagcccggtg cctgagcggg actctggggt tcgaaatgac
     5461 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     5521 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     5581 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     5641 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     5701 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     5761 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     5821 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     5881 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5941 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     6001 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     6061 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     6121 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     6181 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     6241 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     6301 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6361 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6421 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6481 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6541 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     6601 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     6661 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6721 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     6781 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     6841 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     6901 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     6961 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     7021 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     7081 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     7141 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     7201 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     7261 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     7321 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     7381 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     7441 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     7501 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     7561 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     7621 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     7681 tg
//
