LOCUS       sequence-433018-        7511 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Encoding dominant negative Rac1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7511)
  TITLE     Collective cell migration is coordinated by front-biased activation 
            of the feed-forward loop consisting of Ras, Rab5, and Rac1.
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7511)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pPBpuro-Rac1T17N
FEATURES             Location/Qualifiers
     source          1..7511
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     893..915
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     977..996
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(1045..1092,1093..1095,1096..1623)
                     /codon_start=1
                     /product="dominant negative mutant of human Rac family 
                     small GTPase 1"
                     /label=Rac1 (T17N)
                     /translation="MQAIKCVVVGDGAVGKNCLLISYTTNAFPGEYIPTVFDNYSANVM
                     VDGKPVNLGLWDTAGQEDYDRLRPLSYPQTDVFLICFSLVSPASFENVRAKWYPEVRHH
                     CPNTPIILVGTKLDLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQR
                     GLKTVFDEAIRAVLCPPPVKKRKRKCLLL"
     CDS             1045..1623
                     /codon_start=1
                     /product="human Rac family small GTPase 1"
                     /label=Rac1
                     /translation="MQAIKCVVVGDGAVGKNCLLISYTTNAFPGEYIPTVFDNYSANVM
                     VDGKPVNLGLWDTAGQEDYDRLRPLSYPQTDVFLICFSLVSPASFENVRAKWYPEVRHH
                     CPNTPIILVGTKLDLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQR
                     GLKTVFDEAIRAVLCPPPVKKRKRKCLLL"
     misc_feature    1701..2250
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1845..1862)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2072..2091
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2253..2852
                     /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(2253..2272)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2749..2769
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2879..2896)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2885..3109
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3156..3172)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3180..3196
                     /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(3204..3210,3211..3228,3229..3234))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3249..3270
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   3504..3566
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3607..3627)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3607..3625)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3646..3662)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3670..3686
                     /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(3694..3700,3701..3718,3719..3724))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3739..3760
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3877..3894)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4048..4636)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4128..4147)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4807..5598,5599..5667))
                     /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     5430..5449
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5668..5772)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5798..6253)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5935..5956)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6147..6166
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6380..6402
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6394..6411
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6395..6411
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6421..6440
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6421..6439
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   6469..6503
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        6806..7185
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7187..7463
                     /label=chicken beta-actin promoter
     intron          join(7464..7511,1..969)
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
ORIGIN
        1 tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac
       61 ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg
      121 tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg
      181 gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc
      241 ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg
      301 agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc
      361 gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa
      421 ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc
      481 cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg
      541 ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc
      601 cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg
      661 tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg
      721 cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa
      781 atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct
      841 cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc
      901 ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt
      961 ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa
     1021 ttgggatccg aattcctcga ggggatgcag gccatcaagt gtgtggtggt gggagacgga
     1081 gctgtaggta aaaattgcct actgatcagt tacacaacca atgcatttcc tggagaatat
     1141 atccctactg tctttgacaa ttattctgcc aatgttatgg tagatggaaa accggtgaat
     1201 ctgggcttat gggatacagc tggacaagaa gattatgaca gattacgccc cctatcctat
     1261 ccgcaaacag atgtgttctt aatttgcttt tcccttgtga gtcctgcatc atttgaaaat
     1321 gtccgtgcaa agtggtatcc tgaggtgcgg caccactgtc ccaacactcc catcatccta
     1381 gtgggaacta aacttgatct tagggatgat aaagacacga tcgagaaact gaaggagaag
     1441 aagctgactc ccatcaccta tccgcagggt ctagccatgg ctaaggagat tggtgccgta
     1501 aaatacctgg agtgctcggc gctcacacag cgaggcctca agacagtgtt tgacgaagcg
     1561 atccgagcag tcctctgccc gcctcccgtg aagaagagga agagaaaatg cctgctgttg
     1621 taagcggccg ctctagagtc aactaactta agctagcaac ggtttccctc tagcgggatc
     1681 aattccgccc ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc
     1741 gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa
     1801 acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat
     1861 gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac
     1921 aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg
     1981 cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt
     2041 tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg
     2101 gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac
     2161 atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga
     2221 cgtggttttc ctttgaaaaa cacgataata ccatgaccga gtacaagccc acggtgcgcc
     2281 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact
     2341 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc
     2401 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     2461 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     2521 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     2581 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     2641 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     2701 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     2761 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     2821 cctggtgcat gacccgcaag cccggtgcct gatcgatgat ctagagctcg ctgatcagcc
     2881 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     2941 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     3001 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     3061 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     3121 gaaagaacct gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3181 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3241 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3301 tcgggaaacc tgtcgtgcca gcggatctta aaagttttgt tactttatag aagaaatttt
     3361 gagtttttgt ttttttttaa taaataaata aacataaata aattgtttgt tgaatttatt
     3421 attagtatgt aagtgtaaat ataataaaac ttaatatcta ttcaaattaa taaataaacc
     3481 tcgatataca gaccgataaa acacatgcgt caattttacg catgattatc tttaacgtac
     3541 gtcacaatat gattatcttt ctagggttaa tctagtatac gcgtttaatt aatccagctt
     3601 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     3661 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     3721 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     3781 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     3841 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3901 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3961 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4021 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4081 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4141 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4201 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4261 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4321 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4381 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4441 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4501 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4561 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4621 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4681 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4741 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4801 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4861 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4921 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4981 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5041 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5101 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5161 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5221 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5281 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5341 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5401 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5461 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5521 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5581 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5641 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5701 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5761 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg
     5821 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     5881 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     5941 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     6001 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     6061 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     6121 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     6181 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     6241 ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     6301 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     6361 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg
     6421 taatacgact cactataggg cgaattgggg cgcgccattc tagattaacc ctagaaagat
     6481 agtctgcgta aaattgacgc atgcattctt gaaatattgc tctctctttc taaatagcgc
     6541 gaatccgtcg ctgtgcattt aggacatctc agtcgccgct tggagctccc gtgaggcgtg
     6601 cttgtcaatg cggtaagtgt cactgatttt gaactataac gaccgcgtga gtcaaaatga
     6661 cgcatgatta tcttttacgt gacttttaag atttaactca tacgataatt atattgttat
     6721 ttcatgttct acttacgtga taacttatta tatatatatt ttcttgttat agatatcaac
     6781 tagaatgcta gcatgggccc atctcgacat tgattattga ctagttatta atagtaatca
     6841 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     6901 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     6961 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     7021 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     7081 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     7141 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtcga ggtgagcccc
     7201 acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt
     7261 attttttaat tattttgtgc agcgatgggg gcgggggggg gggggggcgc gcgccaggcg
     7321 gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc
     7381 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
     7441 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
     7501 ccgccgccgc c
//
