LOCUS       sequence-227753-        7323 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7323)
  AUTHORS   Pertz O, Hodgson L, Klemke RL, Hahn KM
  TITLE     Spatiotemporal dynamics of RhoA activity in migrating cells.
  JOURNAL   Nature. 2006 Apr 20. 440(7087):1069-72.
  PUBMED    16547516
REFERENCE   2  (bases 1 to 7323)
  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: "Nature. 
            2006 Apr 20. 440(7087):1069-72."
COMMENT     Sequence Label: pTriEx-RhoA FLARE.sc Biosensor WT
FEATURES             Location/Qualifiers
     source          1..7323
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        306..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     711..733
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     795..814
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     856..875
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        856..874
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    875..899
                     /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        915..1024
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     CDS             1045..1062
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(1327..1329,1330..1332,1333..2043)
                     /codon_start=1
                     /product="cyan variant of GFP"
                     /label=CFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2116..2118,2119..2121,2122..2832)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2839..3420
                     /codon_start=1
                     /product="human Rho family small GTPase RhoA"
                     /label=RhoA
                     /translation="MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVAD
                     IEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVK
                     HFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKT
                     KDGVREVFEMATRAALQARRGKKKSGCLVL"
     CDS             3430..3453
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(3543..3562)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3608..3663
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3662..3681)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3751..3798
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     3811..4516
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           3899..4271
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(3928..4516)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4525..4558)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(4726..5313)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4805..4824)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5326..6117,6118..6186))
                     /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     5949..5968
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     misc_recomb     6429..7256
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             6429..6553
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(6591..7196)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtccgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta cgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catgctgatg cggttttggc agtacaccaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagacgtcg tttagtgaac cgtcagatca ctagatgctt
      541 tattgcggta gtttatcaca gttaaattgc taacgccagt ctcgaactta acgtgcagaa
      601 gttggtcgtg aggcactggg caggtaagta tcgggccctt tgtgcggggg gagcggctcg
      661 gggctgtccg cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt
      721 ctggcgtgtg accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt
      781 cctacagctc ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt
      841 ggatcggacc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
      901 cggagttaat ccgggacctt taattcaacc caacacaata tattatagtt aaataagaat
      961 tattatcaaa tcatttgtat attaattaaa atactatact gtaaattaca ttttatttac
     1021 aatcaaagga gatataccat ggcacaccat caccaccatc acggtagtgg catcctggag
     1081 gacctcaata tgctctacat ccggcagatg gcactcagcc tggaggacac agagctgcag
     1141 aggaaactag atcatgagat ccggatgagg gatggggcct gcaagctgct ggcagcctgc
     1201 tcccagcgag agcaggctct ggaagccacc aagagcctgc tggtgtgcaa cagccgtatt
     1261 ctcagctaca tgggtgagct gcagcggcga aaggaggccc aggtgctgga gaagacaggc
     1321 gggggaatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     1381 ctggacggcg acgtaaacgg ccacaggttc agcgtgtccg gcgagggcga gggcgatgcc
     1441 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     1501 cccaccctcg tgaccaccct gacctggggc gtgcagtgct tcagccgcta ccccgaccac
     1561 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgtacc
     1621 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     1681 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     1741 gggcacaagc tggagtacaa ctacatcagc cacaacgtct atatcaccgc cgacaagcag
     1801 aagaacggca tcaaggccca cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     1861 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     1921 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     1981 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     2041 aagggatcta cttctggttc tggtaaacct ggttctggtg aaggttctac taaaggtggt
     2101 agtggcgcgg ccgctatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     2161 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     2221 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     2281 gtgccctggc ccaccctcgt gaccaccttc ggctacggcc tgatgtgctt cgcccgctac
     2341 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     2401 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     2461 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     2521 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     2581 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     2641 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     2701 ctgcccgaca accactacct gagctaccag tccgccctga gcaaagaccc caacgagaag
     2761 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     2821 gagctgtaca aggggggaat ggctgccatc cggaagaaac tggtgattgt tggtgatgga
     2881 gcctgtggaa agacatgctt gctcatagtc ttcagcaagg accagttccc agaggtgtat
     2941 gtgcccacag tgtttgagaa ctatgtggca gatatcgagg tggatggaaa gcaggtagag
     3001 ttggctttgt gggacacagc tgggcaggaa gattatgatc gcctgaggcc cctctcctac
     3061 ccagataccg atgttatact gatgtgtttt tccatcgaca gccctgatag tttagaaaac
     3121 atcccagaaa agtggacccc agaagtcaag catttctgtc ccaacgtgcc catcatcctg
     3181 gttgggaata agaaggatct tcggaatgat gagcacacaa ggcgggagct agccaagatg
     3241 aagcaggagc cggtgaaacc tgaagaaggc agagatatgg caaacaggat tggcgctttt
     3301 gggtacatgg agtgttcagc aaagaccaaa gatggagtga gagaggtttt tgaaatggct
     3361 acgagagctg ctctgcaagc tagacgtggg aagaaaaaat ctggttgcct tgtcttgtga
     3421 taactcgagc accaccatca ccatcaccat cactaagtga ttaacctcag gtgcaggctg
     3481 cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc
     3541 gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact
     3601 tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc
     3661 tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt
     3721 tagagtttgg caacatatgc ccatatgtaa ctagcataac cccttggggc ctctaaacgg
     3781 gtcttgaggg gttttttgct gaaagcatgc ggaggaaatt ctccttgaag tttccctggt
     3841 gttcaaagta aaggagtttg caccagacgc acctctgttc actggtccgg cgtattaaaa
     3901 cacgatacat tgttattagt acatttatta agcgctagat tctgtgcgtt gttgatttac
     3961 agacaattgt tgtacgtatt ttaataattc attaaattta taatctttag ggtggtatgt
     4021 tagagcgaaa atcaaatgat tttcagcgtc tttatatctg aatttaaata ttaaatcctc
     4081 aatagatttg taaaataggt ttcgattagt ttcaaacaag ggttgttttt ccgaaccgat
     4141 ggctggacta tctaatggat tttcgctcaa cgccacaaaa cttgccaaat cttgtagcag
     4201 caatctagct ttgtcgatat tcgtttgtgt tttgttttgt aataaaggtt cgacgtcgtt
     4261 caaaatatta tgcgcttttg tatttctttc atcactgtcg ttagtgtaca attgactcga
     4321 cgtaaacacg ttaaatagag cttggacata tttaacatcg ggcgtgttag ctttattagg
     4381 ccgattatcg tcgtcgtccc aaccctcgtc gttagaagtt gcttccgaag acgattttgc
     4441 catagccaca cgacgcctat taattgtgtc ggctaacacg tccgcgatca aatttgtagt
     4501 tgagcttttt ggaattgcga tcgcataact tcgtatagca tacattatac gaagttataa
     4561 gctcggaacg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4621 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4681 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4741 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4801 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4861 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4921 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4981 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5041 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5101 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5161 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5221 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5281 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgttacc aatgcttaat
     5341 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5401 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5461 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5521 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5581 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5641 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5701 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5761 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5821 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5881 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5941 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6001 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6061 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6121 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6181 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgtc
     6241 cgcgcgtttc ctgcatcttt taatcaaatc ccaagatgtg tataaacgcg ccggtatgta
     6301 caggaagagg tttatactaa actgttacat tgcaaacgtg gtttcgtgtg ccaagtgtga
     6361 aaaccgatgt ttaatcaagg ctctgacgca tttctacaac cacgactcca agtgtgtggg
     6421 tgaagtcatg catcttttaa tcaaatccca agatgtgtat aaaccaccaa actgccaaaa
     6481 aatgaaaact gtcgacaagc tctgtccgtt tgctggcaac tgcaagggtc tcaatcctat
     6541 ttgtaattat tgaataataa aacaattata aatgtcaaat ttgtttttta ttaacgatac
     6601 aaaccaaacg caacaagaac atttgtagta ttatctataa ttgaaaacgc gtagttataa
     6661 tcgctgaggt aatatttaaa atcattttca aatgattcac agttaatttg cgacaatata
     6721 attttatttt cacataaact agacgccttg tcgtcttctt cttcgtattc cttctctttt
     6781 tcatttttct cttcataaaa attaacatag ttattatcgt atccatatat gtatctatcg
     6841 tatagagtaa attttttgtt gtcataaata tatatgtctt ttttaatggg gtgtatagta
     6901 ccgctgcgca tagtttttct gtaatttaca acagtgctat tttctggtag ttcttcggag
     6961 tgtgttgctt taattattaa atttatataa tcaatgaatt tgggatcgtc ggttttgtac
     7021 aatatgttgc cggcatagta cgcagcttct tctagttcaa ttacaccatt ttttagcagc
     7081 accggattaa cataactttc caaaatgttg tacgaaccgt taaacaaaaa cagttcacct
     7141 cccttttcta tactattgtc tgcgagcagt tgtttgttgt taaaaataac agccattgta
     7201 atgagacgca caaactaata tcacaaactg gaaatgtcta tcaatatata gttgctgatg
     7261 gccggcctat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     7321 ccg
//
