LOCUS       sequence-448473-        8642 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express mEGFP-fusion protein of ROCK2 fragment (1-941).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8642)
  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 8642)
  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-ROCK2-N (1-941)
FEATURES             Location/Qualifiers
     source          1..8642
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(905..907,908..910,911..1621)
                     /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(950..971)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1211..1230)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1558..1579
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4482..4499)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4488..4712
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4758..5186
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4845..4864)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5055..5076
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5195..5215)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5200..5529
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5380..5515
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5442..5461
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5542..6141
                     /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(5542..5561)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6038..6058
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6315..6436
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6352..6371)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6406..6425
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6485..6501)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6485..6501)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6498..6520)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6509..6525
                     /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(6533..6539,6540..6557,6558..6563))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6578..6599
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6716..6733)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6887..7475)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6967..6986)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7646..8437,8438..8506))
                     /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     8269..8288
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8507..8611)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 aaccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
      961 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     1021 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     1081 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     1141 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     1201 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1261 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1321 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1381 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1441 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1501 ccactacctg agcacccagt ccaagctgag caaagacccc aacgagaagc gcgatcacat
     1561 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1621 gatgagccgg cccccgccga cggggaaaat gcccggcgcc cccgagaccg cgccggggga
     1681 cggggcaggc gcgagccgcc agaggaagct ggaggcgctg atccgagacc ctcgctcccc
     1741 catcaacgtg gagagcttgc tggatggctt aaattccttg gtccttgatt tagattttcc
     1801 tgctttgagg aaaaacaaga acatagataa tttcttaaat agatatgaga aaattgtgaa
     1861 aaaaatcaga ggtctacaga tgaaggcaga agactatgat gttgtaaaag ttattggaag
     1921 aggtgctttt ggtgaagtgc agttggttcg tcacaaggca tcgcagaagg tttatgctat
     1981 gaagcttctt agtaagtttg aaatgataaa aagatcagat tctgcctttt tttgggaaga
     2041 aagagatatt atggcctttg ccaatagccc ctgggtggtt cagctttttt atgcctttca
     2101 agatgatagg tatctgtaca tggtaatgga gtacatgcct ggtggagacc ttgtaaacct
     2161 tatgagtaat tatgatgtgc ctgaaaaatg ggccaaattt tacactgctg aagttgttct
     2221 tgctctggat gcaatacact ccatgggttt aatacacaga gatgtgaagc ctgacaacat
     2281 gctcttggat aaacatggac atctaaaatt agcagatttt ggcacgtgta tgaagatgga
     2341 tgaaacaggc atggtacatt gtgatacagc agttggaaca ccggattata tatcacctga
     2401 ggttctgaaa tcacaagggg gtgatggttt ctatgggcga gaatgtgatt ggtggtctgt
     2461 aggtgttttc ctttatgaga tgctagtggg ggatactcca ttttatgcgg attcacttgt
     2521 aggaacatat agcaaaatta tggatcataa gaattcactg tgtttccctg aagatgcaga
     2581 aatttccaaa catgcaaaga atctcatctg tgctttctta acagataggg aggtacgact
     2641 tgggagaaat ggggtggaag aaatcagaca gcatcctttc tttaagaatg atcagtggca
     2701 ttgggataac ataagagaaa cggcagctcc tgtagtacct gaactcagca gtgacataga
     2761 cagcagcaat ttcgatgaca ttgaagatga caaaggagat gtagaaacct tcccaattcc
     2821 taaagctttt gttggaaatc agctgccttt catcggattt acctactata gagaaaattt
     2881 attattaagt gactctccat cttgtagaga aactgattcc atacaatcaa ggaaaaatga
     2941 agaaagtcaa gagattcaga aaaaactgta tacattagaa gaacatctta gcaatgagat
     3001 gcaagccaaa gaggaactgg aacagaagtg caaatctgtt aatactcgcc tagaaaaaac
     3061 agcaaaggag ctagaagagg agattacctt acggaaaagt gtggaatcag cattaagaca
     3121 gttagaaaga gaaaaggcgc ttcttcagca caaaaatgca gaatatcaga ggaaagctga
     3181 tcatgaagca gacaaaaaac gaaatttgga aaatgatgtt aacagcttaa aagatcaact
     3241 tgaagatttg aaaaaaagaa atcaaaactc tcaaatatcc actgagaaag tgaatcaact
     3301 ccagagacaa ctggatgaaa ccaatgcttt actgcgaaca gagtctgata ctgcagcccg
     3361 gttaaggaaa acccaggcag aaagttcaaa acagattcag cagctggaat ctaacaatag
     3421 agatctacaa gataaaaact gcctgctgga gactgccaag ttaaaacttg aaaaggaatt
     3481 tatcaatctt cagtcagctc tagaatctga aaggagggat cgaacccatg gatcagagat
     3541 aattaatgat ttacaaggta gaatatgtgg cctagaagaa gatttaaaga acggcaaaat
     3601 cttactagcg aaagtagaac tggagaagag acaacttcag gagagattta ctgatttgga
     3661 aaaggaaaaa agcaacatgg aaatagatat gacataccaa ctaaaagtta tacagcagag
     3721 cctagaacaa gaagaagctg aacataaggc cacaaaggca cgactagcag ataaaaataa
     3781 gatctatgag tccatcgaag aagccaaatc agaagccatg aaagaaatgg agaagaagct
     3841 cttggaggaa agaactttaa aacagaaagt ggagaaccta ttgctagaag ctgagaaaag
     3901 atgttctcta ttagactgtg acctcaaaca gtcacagcag aaaataaatg agctccttaa
     3961 acagaaagat gtgctaaatg aggatgttag aaacctgaca ttaaaaatag agcaagaaac
     4021 tcagaagcgc tgccttacac aaaatgacct gaagatgcaa acacaacagg ttaacacact
     4081 aaaaatgtca gaaaagcagt taaagcaaga aaataaccat ctcatggaaa tgaaaatgaa
     4141 cttggaaaaa caaaatgctg aacttcgaaa agaacgtcag gatgcagatg ggcaaatgaa
     4201 agagctccag gatcagctcg aagcagaaca gtatttctca accctttata aaacacaagt
     4261 tagggagctt aaagaagaat gtgaagaaaa gaccaaactt ggtaaagaat tgcagcagaa
     4321 gaaacaggaa ttacaggatg aacgggactc tttggctgcc caactggaga tcaccttgac
     4381 caaagcagat tctgagcaac tggctcgttc aattgctgaa gaacaatatt ctgatttgga
     4441 ataactcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
     4501 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     4561 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     4621 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     4681 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     4741 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     4801 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     4861 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     4921 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     4981 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     5041 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     5101 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     5161 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     5221 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     5281 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     5341 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     5401 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     5461 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     5521 ttttgcaaaa agctcccaac catgaccgag tacaagccca cggtgcgcct cgccacccgc
     5581 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     5641 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     5701 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
     5761 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     5821 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
     5881 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac
     5941 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
     6001 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg
     6061 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     6121 acccgcaagc ccggtgcctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     6181 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     6241 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     6301 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     6361 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     6421 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     6481 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     6541 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     6601 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     6661 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     6721 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     6781 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     6841 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     6901 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     6961 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     7021 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     7081 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     7141 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     7201 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     7261 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     7321 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     7381 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     7441 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     7501 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     7561 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     7621 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     7681 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     7741 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     7801 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     7861 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     7921 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     7981 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     8041 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     8101 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     8161 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     8221 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     8281 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     8341 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     8401 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     8461 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     8521 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     8581 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     8641 tc
//
