LOCUS       sequence-160183-        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 T19N
FEATURES             Location/Qualifiers
     source          1..7323
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             7..24
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(289..291,292..294,295..1005)
                     /codon_start=1
                     /product="cyan variant of GFP"
                     /label=CFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1078..1080,1081..1083,1084..1794)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSTMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1801..1854,1855..1857,1858..2382)
                     /codon_start=1
                     /product="dominant negative mutant of human Rho family 
                     small GTPase RhoA"
                     /label=RhoA (T19N)
                     /translation="MAAIRKKLVIVGDGACGKNCLLIVFSKDQFPEVYVPTVFENYVAD
                     IEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVK
                     HFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKT
                     KDGVREVFEMATRAALQARRGKKKSGCLVL"
     CDS             2392..2415
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2505..2524)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2570..2625
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2624..2643)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      2713..2760
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     2773..3478
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           2861..3233
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(2890..3478)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(3487..3520)
                     /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(3688..4275)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3767..3786)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4288..5079,5080..5148))
                     /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     4911..4930
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     misc_recomb     5391..6218
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             5391..5515
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(5553..6158)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        6286..6589
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6591..6793
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6744..6764
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6996..7018
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     7080..7099
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     7141..7160
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7141..7159
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7160..7184
                     /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        7200..7309
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
ORIGIN
        1 atggcacacc atcaccacca tcacggtagt ggcatcctgg aggacctcaa tatgctctac
       61 atccggcaga tggcactcag cctggaggac acagagctgc agaggaaact agatcatgag
      121 atccggatga gggatggggc ctgcaagctg ctggcagcct gctcccagcg agagcaggct
      181 ctggaagcca ccaagagcct gctggtgtgc aacagccgta ttctcagcta catgggtgag
      241 ctgcagcggc gaaaggaggc ccaggtgctg gagaagacag gcgggggaat ggtgagcaag
      301 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
      361 ggccacaggt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
      421 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
      481 ctgacctggg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
      541 ttcaagtccg ccatgcccga aggctacgtc caggagcgta ccatcttctt caaggacgac
      601 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
      661 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
      721 aactacatca gccacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc
      781 cacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
      841 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
      901 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
      961 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggatc tacttctggt
     1021 tctggtaaac ctggttctgg tgaaggttct actaaaggtg gtagtggcgc ggccgctatg
     1081 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1141 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1201 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1261 gtgaccacct tcggctacgg cctgatgtgc ttcgcccgct accccgacca catgaagcag
     1321 cacgacttct tcaagtccac catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     1381 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1441 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1501 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     1561 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     1621 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     1681 ctgagctacc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1741 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggggga
     1801 atggctgcca tccggaagaa actggtgatt gttggtgatg gagcctgtgg aaagaactgc
     1861 ttgctcatag tcttcagcaa ggaccagttc ccagaggtgt atgtgcccac agtgtttgag
     1921 aactatgtgg cagatatcga ggtggatgga aagcaggtag agttggcttt gtgggacaca
     1981 gctgggcagg aagattatga tcgcctgagg cccctctcct acccagatac cgatgttata
     2041 ctgatgtgtt tttccatcga cagccctgat agtttagaaa acatcccaga aaagtggacc
     2101 ccagaagtca agcatttctg tcccaacgtg cccatcatcc tggttgggaa taagaaggat
     2161 cttcggaatg atgagcacac aaggcgggag ctagccaaga tgaagcagga gccggtgaaa
     2221 cctgaagaag gcagagatat ggcaaacagg attggcgctt ttgggtacat ggagtgttca
     2281 gcaaagacca aagatggagt gagagaggtt tttgaaatgg ctacgagagc tgctctgcaa
     2341 gctagacgtg ggaagaaaaa atctggttgc cttgtcttgt gataactcga gcaccaccat
     2401 caccatcacc atcactaagt gattaacctc aggtgcaggc tgcctatcag aaggtggtgg
     2461 ctggtgtggc caatgccctg gctcacaaat accactgaga tcgatctttt tccctctgcc
     2521 aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa
     2581 tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat
     2641 gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat
     2701 gcccatatgt aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     2761 ctgaaagcat gcggaggaaa ttctccttga agtttccctg gtgttcaaag taaaggagtt
     2821 tgcaccagac gcacctctgt tcactggtcc ggcgtattaa aacacgatac attgttatta
     2881 gtacatttat taagcgctag attctgtgcg ttgttgattt acagacaatt gttgtacgta
     2941 ttttaataat tcattaaatt tataatcttt agggtggtat gttagagcga aaatcaaatg
     3001 attttcagcg tctttatatc tgaatttaaa tattaaatcc tcaatagatt tgtaaaatag
     3061 gtttcgatta gtttcaaaca agggttgttt ttccgaaccg atggctggac tatctaatgg
     3121 attttcgctc aacgccacaa aacttgccaa atcttgtagc agcaatctag ctttgtcgat
     3181 attcgtttgt gttttgtttt gtaataaagg ttcgacgtcg ttcaaaatat tatgcgcttt
     3241 tgtatttctt tcatcactgt cgttagtgta caattgactc gacgtaaaca cgttaaatag
     3301 agcttggaca tatttaacat cgggcgtgtt agctttatta ggccgattat cgtcgtcgtc
     3361 ccaaccctcg tcgttagaag ttgcttccga agacgatttt gccatagcca cacgacgcct
     3421 attaattgtg tcggctaaca cgtccgcgat caaatttgta gttgagcttt ttggaattgc
     3481 gatcgcataa cttcgtatag catacattat acgaagttat aagctcggaa cgctgcgctc
     3541 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3601 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3661 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgccccctga cgagcatcac
     3721 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     3781 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     3841 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     3901 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     3961 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4021 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4081 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4141 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4201 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4261 aaaaaaggat ctcaagaaga tcctttgtta ccaatgctta atcagtgagg cacctatctc
     4321 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4381 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4441 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4501 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4561 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4621 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4681 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4741 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4801 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4861 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     4921 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     4981 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5041 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5101 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5161 tcaatattat tgaagcattt atcagggtta ttgtctcatg tccgcgcgtt tcctgcatct
     5221 tttaatcaaa tcccaagatg tgtataaacg cgccggtatg tacaggaaga ggtttatact
     5281 aaactgttac attgcaaacg tggtttcgtg tgccaagtgt gaaaaccgat gtttaatcaa
     5341 ggctctgacg catttctaca accacgactc caagtgtgtg ggtgaagtca tgcatctttt
     5401 aatcaaatcc caagatgtgt ataaaccacc aaactgccaa aaaatgaaaa ctgtcgacaa
     5461 gctctgtccg tttgctggca actgcaaggg tctcaatcct atttgtaatt attgaataat
     5521 aaaacaatta taaatgtcaa atttgttttt tattaacgat acaaaccaaa cgcaacaaga
     5581 acatttgtag tattatctat aattgaaaac gcgtagttat aatcgctgag gtaatattta
     5641 aaatcatttt caaatgattc acagttaatt tgcgacaata taattttatt ttcacataaa
     5701 ctagacgcct tgtcgtcttc ttcttcgtat tccttctctt tttcattttt ctcttcataa
     5761 aaattaacat agttattatc gtatccatat atgtatctat cgtatagagt aaattttttg
     5821 ttgtcataaa tatatatgtc ttttttaatg gggtgtatag taccgctgcg catagttttt
     5881 ctgtaattta caacagtgct attttctggt agttcttcgg agtgtgttgc tttaattatt
     5941 aaatttatat aatcaatgaa tttgggatcg tcggttttgt acaatatgtt gccggcatag
     6001 tacgcagctt cttctagttc aattacacca ttttttagca gcaccggatt aacataactt
     6061 tccaaaatgt tgtacgaacc gttaaacaaa aacagttcac ctcccttttc tatactattg
     6121 tctgcgagca gttgtttgtt gttaaaaata acagccattg taatgagacg cacaaactaa
     6181 tatcacaaac tggaaatgtc tatcaatata tagttgctga tggccggcct attaatagta
     6241 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     6301 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     6361 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     6421 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagtc cgccccctat
     6481 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttacggga
     6541 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgc tgatgcggtt
     6601 ttggcagtac accaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     6661 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     6721 tcgtaataac cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     6781 tataagcaga cgtcgtttag tgaaccgtca gatcactaga tgctttattg cggtagttta
     6841 tcacagttaa attgctaacg ccagtctcga acttaacgtg cagaagttgg tcgtgaggca
     6901 ctgggcaggt aagtatcggg ccctttgtgc ggggggagcg gctcggggct gtccgcgggg
     6961 ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg
     7021 cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg
     7081 caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattggatc ggaccgaaat
     7141 taatacgact cactataggg gaattgtgag cggataacaa ttccccggag ttaatccggg
     7201 acctttaatt caacccaaca caatatatta tagttaaata agaattatta tcaaatcatt
     7261 tgtatattaa ttaaaatact atactgtaaa ttacatttta tttacaatca aaggagatat
     7321 acc
//
