LOCUS       sequence-223086-        5291 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 5291)
  TITLE     Bokoch Lab GTPases
REFERENCE   2  (bases 1 to 5291)
  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"
COMMENT     Sequence Label: pRK5-myc-RhoA-T19N
FEATURES             Location/Qualifiers
     source          1..5291
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    132..153
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(168..173,174..191,192..198)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    206..222
                     /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)."
     primer_bind     211..233
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     230..246
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     230..246
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        275..654
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        655..858
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     809..829
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     855..879
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        1089..1107
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1089..1106
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     1120..1139
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      1186..1195
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1195..1224
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             join(1231..1284,1285..1287,1288..1812)
                     /codon_start=1
                     /product="dominant negative mutant of human Rho family 
                     small GTPase RhoA"
                     /label=RhoA (T19N)
                     /translation="MAAIRKKLVIVGDGACGKNCLLIVFSKDQFPEVYVPTVFENYVAD
                     IEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVK
                     HFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKT
                     KDGVREVFEMATRAALQARRGKKKSGCLVL"
     regulatory      1852..1861
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1865..1999
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1902..1921)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1956..1975
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2068..2425
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2068..2259
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2091..2111)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2276..2411
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2445..2462)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2445..2461)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2454..2476)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2674..3129
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2761..2780)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2971..2992
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3146..3165)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3265..3287
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3325..3343)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3411..3515
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3516..3584,3585..4376)
                     /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     complement(3734..3753)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4547..5135
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5036..5055
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(5289..5291,1..15)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
       61 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
      121 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
      181 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
      241 tatgacatga ttacgaatta attcgagctc gcccgacatt gattattgac tagttattaa
      301 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
      361 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
      421 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
      481 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
      541 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
      601 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
      661 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
      721 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
      781 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
      841 gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc
      901 tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccg ggaacggtgc
      961 attggaacgc ggattccccg tgccaagagt gacgtaagta ccgcctatag agtctatagg
     1021 cccaccccct tggcttcgtt agaacgcggc tacaattaat acataacctt atgtatcata
     1081 cacatacgat ttaggtgaca ctatagaata acatccactt tgcctttctc tccacaggtg
     1141 tccactccca ggtccaactg cacctcggtt ctatcgattg aattggccac catggagcag
     1201 aagctgatct ccgaggagga cctgggatcc atggctgcca tccggaagaa actggtgatt
     1261 gttggtgatg gagcctgtgg aaagaactgc ttgctcatag tcttcagcaa ggaccagttc
     1321 ccagaggtgt atgtgcccac agtgtttgag aactatgtgg cagatatcga ggtggatgga
     1381 aagcaggtag agttggcttt gtgggacaca gctgggcagg aagattatga tcgcctgagg
     1441 cccctctcct acccagatac cgatgttata ctgatgtgtt tttccatcga cagccctgat
     1501 agtttagaaa acatcccaga aaagtggacc ccagaagtca agcatttctg tcccaacgtg
     1561 cccatcatcc tggttgggaa taagaaggat cttcggaatg atgagcacac aaggcgggag
     1621 ctagccaaga tgaagcagga gccggtgaaa cctgaagaag gcagagatat ggcaaacagg
     1681 attggcgctt ttgggtacat ggagtgttca gcaaagacca aagatggagt gagagaggtt
     1741 tttgaaatgg ctacgagagc tgctctgcaa gctagacgtg ggaagaaaaa atctggttgc
     1801 cttgtcttgt gaggatccgc ggccgcgcta gcgaattcct gcagaagctt ggccgccatg
     1861 gcccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     1921 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     1981 atcttatcat gtctggatcg ggaattaatt cggcgcagca ccatggcctg aaataacctc
     2041 tgaaagagga acttggttag gtaccttctg aggcggaaag aaccagctgt ggaatgtgtg
     2101 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2161 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2221 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     2281 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     2341 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     2401 ttttggaggc ctaggctttt gcaaaaagct gttaacagct tggcactggc cgtcgtttta
     2461 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     2521 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     2581 cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     2641 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     2701 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     2761 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2821 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     2881 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     2941 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3001 tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     3061 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     3121 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     3181 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3241 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3301 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     3361 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3421 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3481 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     3541 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     3601 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     3661 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     3721 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     3781 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     3841 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     3901 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     3961 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     4021 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     4081 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     4141 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     4201 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     4261 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     4321 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     4381 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     4441 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4501 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4561 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4621 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4681 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4741 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4801 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4861 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4921 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4981 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     5041 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     5101 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     5161 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     5221 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     5281 ccgagcgcag c
//
