LOCUS       sequence-325086-        5169 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of GFP-CaaX (sequence from Hras) in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5169)
  AUTHORS   Hall TE, Martel N, Ariotti N, Xiong Z, Lo HP, Ferguson C, Rae J, Lim
            YW, Parton RG
  TITLE     In vivo cell biological screening identifies an endocytic capture 
            mechanism for T-tubule formation.
  JOURNAL   Nat Commun. 2020 Jul 24;11(1):3711. doi: 10.1038/s41467-020-17486-w.
  PUBMED    32709891
REFERENCE   2  (bases 1 to 5169)
  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"; journalName: "Nat 
            Commun."; date: "2020-07-24"; volume: "11(1):3711. doi"; pages: " 
            10.1038/s41467-020-17486-w"
COMMENT     Sequence Label: EGFP-CaaX(tH) in pCSDEST2
FEATURES             Location/Qualifiers
     source          1..5169
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        38..56
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     38..55
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    111..135
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      136..145
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(142..144,145..147,148..858)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(142..144,145..147,148..858)
                     /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(187..208)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(448..467)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     795..816
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(860..884)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    978..1112
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(1164..1184)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    1224..1358
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1471..1491)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1471..1489)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1510..1526)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1510..1526)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1523..1545)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1534..1550
                     /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(1558..1564,1565..1582,1583..1588))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1603..1624
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1741..1758)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1912..2500)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1992..2011)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2671..3462,3463..3531))
                     /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     3294..3313
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3532..3636)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3662..4117)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3799..3820)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4011..4030
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(4188..5169,1..3)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 aaccgccatt ctgcctgggg acgtcggagc aagcttgatt taggtgacac tatagaatac
       61 aagctacttg ttctttttgc aggatcccat cgattcgaat tcaaggatca acaagtttgt
      121 acaaaaaagc aggctgccac catggtgagc aagggcgagg agctgttcac cggggtggtg
      181 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
      241 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
      301 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
      361 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
      421 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
      481 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
      541 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
      601 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
      661 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
      721 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
      781 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
      841 atggacgagc tgtacaagca cccagctttc ttgtacaaag tgggaaagct gaaccctcct
      901 gatgagagtg gccccggctg catgagctgc aagtgtgtgc tctcctgatc tagaactata
      961 gtgagtcgta ttacgtagat ccagacatga taagatacat tgatgagttt ggacaaacca
     1021 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     1081 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     1141 ttcaggttca gggggaggtg tggcaacttt attatacata gttgcactcg agcctctaga
     1201 actatagtga gtcgtattac gtagatccag acatgataag atacattgat gagtttggac
     1261 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     1321 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     1381 ttatgtttca ggttcagggg gaggtgtggg aggtttttta attcgcggcc gcggcgccaa
     1441 tgcattgggc ccggtaccca gcttttgttc cctttagtga gggttaattg cgcgcttggc
     1501 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     1561 catacgagcc gggagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     1621 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     1681 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     1741 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     1801 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     1861 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     1921 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     1981 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     2041 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     2101 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     2161 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     2221 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     2281 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     2341 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     2401 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     2461 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     2521 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     2581 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     2641 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     2701 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     2761 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     2821 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     2881 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     2941 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     3001 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     3061 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     3121 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     3181 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     3241 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     3301 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     3361 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     3421 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     3481 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     3541 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     3601 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     3661 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     3721 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     3781 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     3841 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     3901 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     3961 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     4021 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     4081 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc
     4141 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagtc gaccgccaat
     4201 tcaatatggc gtatatggac tcatgccaat tcaatatggt ggatctggac ctgtgccaat
     4261 tcaatatggc gtatatggac tcgtgccaat tcaatatggt ggatctggac cccagccaat
     4321 tcaatatggc ggacttggca ccatgccaat tcaatatggc ggacctggca ctgtgccaac
     4381 tggggagggg tctacttggc acggtgccaa gtttgaggag gggtcttggc cctgtgccaa
     4441 gtccgccata ttgaattggc atggtgccaa taatggcggc catattggct atatgccagg
     4501 atcaatatat aggcaatatc caatatggcc ctatgccaat atggctattg gccaggttca
     4561 atactatgta ttggccctat gccatatagt attccatata tgggttttcc tattgacgta
     4621 gatagcccct cccaatgggc ggtcccatat accatatatg gggcttccta ataccgccca
     4681 tagccactcc cccattgacg tcaatggtct ctatatatgg tctttcctat tgacgtcata
     4741 tgggcggtcc tattgacgta tatggcgcct cccccattga cgtcaattac ggtaaatggc
     4801 ccgcctggct caatgcccat tgacgtcaat aggaccaccc accattgacg tcaatgggat
     4861 ggctcattgc ccattcatat ccgttctcac gccccctatt gacgtcaatg acggtaaatg
     4921 gcccacttgg cagtacatca atatctatta atagtaactt ggcaagtaca ttactattgg
     4981 aagtacgcca gggtacattg gcagtactcc cattgacgtc aatggcggta aatggcccgc
     5041 gatggctgcc aagtacatcc ccattgacgt caatggggag gggcaatgac gcaaatgggc
     5101 gttccattga cgtaaatggg cggtaggcgt gcctaatggg aggtctatat aagcaatgct
     5161 cgtttaggg
//
