LOCUS       sequence-204190-        5286 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Control for FRET biosensor analysis - cannot bind downstream 
            effectors.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5286)
  AUTHORS   Petrie RJ, Gavara N, Chadwick RS, Yamada KM
  TITLE     Nonpolarized signaling reveals two distinct modes of 3D cell 
            migration.
  JOURNAL   J Cell Biol. 2012 Apr 30;197(3):439-55. doi: 10.1083/jcb.201201124.
  PUBMED    22547408
REFERENCE   2  (bases 1 to 5286)
  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"; doi: "10.1083/jcb"; 
            journalName: "J Cell Biol"; date: "2012-04-30- 30"; volume: "197"; 
            issue: "3"; pages: "439-55"
COMMENT     Sequence Label: pECFP-Rac1Y40C
FEATURES             Location/Qualifiers
     source          1..5286
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      26..614
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     515..534
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        789..1092
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1093..1296
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1247..1267
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1335..1344
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1341..1343,1344..1346,1347..2057)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1386..1407)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1647..1666)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1994..2015
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2094..2672
                     /codon_start=1
                     /product="human Rac family small GTPase 1"
                     /label=Rac1
                     /translation="MQAIKCVVVGDGAVGKTCLLISYTTNAFPGEYIPTVFDNCSANVM
                     VDGKPVNLGLWDTAGQEDYDRLRPLSYPQTDVFLICFSLVSPASFENVRAKWYPEVRHH
                     CPNTPIILVGTKLDLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQR
                     GLKTVFDEAIRAVLCPPPVKKRKRKCLLL"
     polyA_signal    2802..2923
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2839..2858)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2893..2912
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2930..3385)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3067..3088)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3279..3298
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3412..3516
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3518..3875
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3518..3709
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3541..3561)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3726..3861
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3910..4704
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3910..4704
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3964..3983)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4574..4593
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4892..4911)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4936..4983
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
       61 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
      121 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
      181 tgtagccgta gttaggccac cacttcaaga actctgtatc accgcctaca tacctcgctc
      241 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
      301 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
      361 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
      421 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
      481 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
      541 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
      601 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
      661 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
      721 ccatgcatta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
      781 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
      841 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
      901 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
      961 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     1021 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     1081 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
     1141 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
     1201 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     1261 aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc
     1321 tagcgctacc ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc
     1381 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     1441 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     1501 tgcccgtgcc ctggcccacc ctcgtgacca ccctgacctg gggcgtgcag tgcttcagcc
     1561 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     1621 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     1681 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     1741 acggcaacat cctggggcac aagctggagt acaactacat cagccacaac gtctatatca
     1801 ccgccgacaa gcagaagaac ggcatcaagg ccaacttcaa gatccgccac aacatcgagg
     1861 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     1921 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     1981 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     2041 tggacgagct gtacaagtcc ggactcagat ctcgagctca agcttcgaat tccatgcagg
     2101 ccatcaagtg tgtggtggtg ggagacggag ctgtaggtaa aacttgccta ctgatcagtt
     2161 acacaaccaa tgcatttcct ggagaatata tccctactgt ctttgacaat tgttctgcca
     2221 atgttatggt agatggaaaa ccggtgaacc tgggcttatg ggatacagct ggacaagaag
     2281 attatgacag attacgcccc ctatcctatc cgcaaacaga tgtgttctta atttgctttt
     2341 cccttgtgag tcctgcatca tttgaaaatg tccgtgcaaa gtggtatcct gaggtgcggc
     2401 accactgtcc caacactccc atcatcctag tgggaactaa acttgatctt agggatgata
     2461 aagacacgat cgagaaactg aaggagaaga agctgactcc catcacctat ccgcagggtc
     2521 tagccatggc taaggagatt ggtgccgtaa aatacctgga gtgctcggcg ctcacacagc
     2581 gaggcctcaa gacagtgttt gacgaagcga tccgagcagt cctctgcccg cctcccgtga
     2641 agaagaggaa gagaaaatgc ctgctgttgt aaggatccac cggatctaga taactgatca
     2701 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     2761 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     2821 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     2881 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta aattgtaagc
     2941 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     3001 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     3061 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     3121 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     3181 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     3241 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     3301 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     3361 cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     3421 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3481 tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt
     3541 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3601 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3661 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3721 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3781 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     3841 gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga
     3901 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     3961 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     4021 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     4081 aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     4141 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     4201 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     4261 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     4321 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     4381 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc
     4441 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     4501 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     4561 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     4621 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     4681 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     4741 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     4801 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     4861 agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag
     4921 gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg
     4981 ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc
     5041 attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg
     5101 aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact
     5161 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     5221 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     5281 cagacc
//
