LOCUS       pRK5_mCherry-GFP        5834 bp    DNA     circular SYN 13-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5834)
  TITLE     Spatial spread of Ras activity
REFERENCE   2  (bases 1 to 5834)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 13, 2025 from SnapGene 8.0.2
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pRK5_mCherry-GFP-nb
FEATURES             Location/Qualifiers
     source          1..5834
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     902..971
                     /label=HN181
     regulatory      933..941
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             939..1646
                     /codon_start=1
                     /label=mCherry (full)
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1258..1278)
                     /label=backbone_R
     primer_bind     complement(1622..1671)
                     /label=HN278
     primer_bind     complement(1622..1647)
                     /label=HN182
     primer_bind     1623..1671
                     /label=HN279
     CDS             1647..1667
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1668..1697
                     /codon_start=1
                     /label=GGGGSx2-linker1
                     /translation="GGGGSGGGGS"
     CDS             1698..2081
                     /codon_start=1
                     /label=GFP_nanobody
                     /translation="MAQVQLVESGGRLVQAGDSLRLSCAASGRTFSTSAMAWFRQAPGR
                     EREFVAAITWTVGNTILGDSVKGRFTISRDRAKNTVDLQMDNLEPEDTAVYYCSARSRG
                     YVLSVLRSVDSYDYWGQGTQVTVS"
     CDS             2082..2111
                     /codon_start=1
                     /label=GGGGSx2-linker2
                     /translation="GGGGSGGGGS"
     primer_bind     complement(2092..2136)
                     /label=HN284
     regulatory      2134..2143
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2147..2281
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2184..2203)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2238..2257
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2350..2707
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(2373..2393)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2558..2693
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2620..2639
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(2643..2654)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(2727..2744)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2727..2743)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2736..2758)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2956..3411
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3043..3062)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3253..3274
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3428..3447)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3547..3569
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3607..3625)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3693..3797
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3798..3866,3867..4658)
                     /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(4016..4035)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4829..5417
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5318..5337
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5571..5588
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(5741..5746,5747..5764,5765..5771)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5779..5795
                     /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     5784..5806
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5803..5819
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5803..5819
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc
      901 acctcggttc tatcgattga attcgtaccg gtgccaccat ggtttccaaa ggagaggagg
      961 ataatatggc tataattaaa gagtttatgc ggttcaaggt gcatatggag ggatctgtga
     1021 acggtcacga gttcgaaatc gagggcgaag gcgaggggcg accctatgaa gggacacaga
     1081 cggccaaact gaaggtgacc aagggtggcc ccctgccttt cgcctgggac atcctgtcac
     1141 cccagtttat gtacggctct aaggcttacg ttaagcaccc tgccgatatt ccggactact
     1201 tgaaactgag cttccctgaa gggttcaagt gggaacgggt aatgaatttt gaggatggcg
     1261 gtgtagtcac agttacccag gacagctctc ttcaggacgg agaatttatc tataaggtta
     1321 aacttcgggg cactaacttc ccatcagacg gccccgtgat gcagaagaaa actatgggct
     1381 gggaagccag ctcagaacgc atgtatcccg aggacggagc actgaaggga gaaatcaagc
     1441 agcgactcaa gctgaaggat gggggacatt atgatgctga ggtcaaaacc acctataagg
     1501 ccaagaaacc cgttcagctt cctggggcat acaacgtgaa tatcaaactg gatattacat
     1561 cccataacga agactatacc atcgtggaac agtacgagcg ggccgagggc agacatagca
     1621 cagggggcat ggatgaattg tacaagccta agaagaagag gaaggttggg ggaggcggca
     1681 gtggcggagg gggatctatg gctcaggtgc agctggtgga atctggcggc agactggtgc
     1741 aggccggcga tagcctgaga ctgtcttgtg ccgccagcgg cagaaccttc agcacatctg
     1801 ccatggcctg gttcagacag gcccctggcc gcgagaggga atttgtggcc gccatcacat
     1861 ggaccgtggg caacaccatc ctgggcgaca gcgtgaaggg ccggttcacc atcagccggg
     1921 acagagccaa gaacaccgtg gacctccaga tggacaacct ggaacccgag gacaccgccg
     1981 tgtactactg ctccgccaga tccagaggct acgtgctgtc cgtgctgcgg agcgtggaca
     2041 gctacgatta ttggggccag ggcacccaag tgaccgtgtc tggcggcgga ggaagcggag
     2101 gcggaggatc ttaggtcgac ctgcagaagc ttggccgcca tggcccaact tgtttattgc
     2161 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     2221 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat
     2281 cgggaattaa ttcggcgcag caccatggcc tgaaataacc tctgaaagag gaacttggtt
     2341 aggtaccttc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa
     2401 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     2461 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     2521 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     2581 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     2641 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     2701 ttgcaaaaag ctgttaacag cttggcactg gccgtcgttt tacaacgtcg tgactgggaa
     2761 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     2821 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     2881 tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     2941 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3001 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3061 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3121 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     3181 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3241 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcgggct
     3301 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3361 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     3421 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     3481 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3541 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     3601 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     3661 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     3721 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3781 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     3841 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     3901 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     3961 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     4021 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     4081 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     4141 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     4201 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     4261 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     4321 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     4381 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     4441 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     4501 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     4561 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     4621 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     4681 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     4741 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4801 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4861 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4921 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     4981 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     5041 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5101 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5161 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5221 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5281 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5341 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5401 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5461 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     5521 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     5581 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     5641 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     5701 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     5761 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgaca
     5821 tgattacgaa ttaa
//
