LOCUS       Exported                6680 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA3-EGFP-RhoA-T19N
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6680)
  AUTHORS   Subauste MC, Von Herrath M, Benard V, Chamberlain CE, Chuang TH, Chu
            K, Bokoch GM, Hahn KM
  TITLE     Rho family proteins modulate rapid apoptosis induced by cytotoxic T 
            lymphocytes and Fas.
  JOURNAL   J Biol Chem. 2000 Mar 31. 275(13):9725-33.
  PUBMED    10734125
REFERENCE   2  (bases 1 to 6680)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6680
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        123..502
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        503..706
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     657..677
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     751..770
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        751..769
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      783..792
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             789..1505
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             789..1505
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(834..855)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1095..1114)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1442..1463
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1512..2093
                     /codon_start=1
                     /product="dominant negative mutant of human Rho family 
                     small GTPase RhoA"
                     /label=RhoA (T19N)
                     /translation="MAAIRKKLVIVGDGACGKNCLLIVFSKDQFPEVYVPTVFENYVAD
                     IEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVK
                     HFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKT
                     KDGVREVFEMATRAALQARRGKKKSGCLVL"
     promoter        complement(2121..2139)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2122..2139)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2159..2176)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2165..2389
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2435..2863
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2522..2541)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2732..2753
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2872..2892)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2877..3206
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3057..3192
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3119..3138
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3273..4067
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3327..3346)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3937..3956
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4241..4362
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4278..4297)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4332..4351
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4411..4427)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4411..4427)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4424..4446)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4435..4451
                     /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(4459..4489)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4504..4525
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4642..4659)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4813..5401)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4893..4912)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5572..6432)
                     /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     6195..6214
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6433..6537)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6612..6631)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
       61 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
      121 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
      181 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      241 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      301 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      361 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      421 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      481 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      541 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      601 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      661 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag
      721 agaacccact gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc
      781 ttgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
      841 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
      901 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
      961 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     1021 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     1081 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     1141 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     1201 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     1261 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     1321 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     1381 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     1441 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     1501 acaaggaatt catggctgcc atccggaaga aactggtgat tgttggtgat ggagcctgtg
     1561 gaaagaactg cttgctcata gtcttcagca aggaccagtt cccagaggtg tatgtgccca
     1621 cagtgtttga gaactatgtg gcagatatcg aggtggatgg aaagcaggta gagttggctt
     1681 tgtgggacac agctgggcag gaagattatg atcgcctgag gcccctctcc tacccagata
     1741 ccgatgttat actgatgtgt ttttccatcg acagccctga tagtttagaa aacatcccag
     1801 aaaagtggac cccagaagtc aagcatttct gtcccaacgt gcccatcatc ctggttggga
     1861 ataagaagga tcttcggaat gatgagcaca caaggcggga gctagccaag atgaagcagg
     1921 agccggtgaa acctgaagaa ggcagagata tggcaaacag gattggcgct tttgggtaca
     1981 tggagtgttc agcaaagacc aaagatggag tgagagaggt ttttgaaatg gctacgagag
     2041 ctgctctgca agctagacgt gggaagaaaa aatctggttg ccttgtcttg tgactcgagc
     2101 atgcatctag agggccctat tctatagtgt cacctaaatg ctagagctcg ctgatcagcc
     2161 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     2221 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     2281 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     2341 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     2401 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     2461 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     2521 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2581 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2641 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     2701 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     2761 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     2821 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     2881 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     2941 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     3001 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     3061 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     3121 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
     3181 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
     3241 ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     3301 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     3361 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     3421 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
     3481 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     3541 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     3601 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     3661 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     3721 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     3781 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg
     3841 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     3901 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     3961 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     4021 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     4081 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     4141 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     4201 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     4261 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     4321 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     4381 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     4441 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     4501 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4561 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4621 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4681 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4741 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4801 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4861 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4921 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4981 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     5041 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5101 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     5161 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     5221 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     5281 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5341 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5401 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5461 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5521 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5581 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5641 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5701 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5761 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5821 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5881 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5941 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     6001 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     6061 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     6121 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     6181 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     6241 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     6301 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     6361 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6421 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6481 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6541 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
     6601 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     6661 tgtgtgttgg aggtcgctga
//
