LOCUS       sequence-421724-        7896 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for genetic tracing of mesenchymal Glioblastoma
            cell-state..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7896)
  AUTHORS   Company C, Schmitt MJ, Dramaretska Y, Serresi M, Kertalli S, Jiang 
            B, Yin JA, Aguzzi A, Barozzi I, Gargiulo G
  TITLE     Logical design of synthetic cis-regulatory DNA for genetic tracing 
            of cell identities and state changes.
  JOURNAL   Nat Commun. 2024 Feb 5;15(1):897. doi: 10.1038/s41467-024-45069-6.
  PUBMED    38316783
REFERENCE   2  (bases 1 to 7896)
  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: "2024-02-5"; volume: "15(1):897. doi"; pages: " 
            10.1038/s41467-024-45069-6"
COMMENT     Sequence Label: pPB-CLGT3-d2EGFP_CMV-mCherry
FEATURES             Location/Qualifiers
     source          1..7896
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   5..39
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    316..336
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    1396..1420
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1421..1430
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1427..1429,1430..1432,1433..2143,2144..2149,
                     2150..2272)
                     /codon_start=1
                     /product="EGFP destabilized by fusion to residues 422-461 
                     of mouse ornithine decarboxylase, giving an in vivo 
                     half-life of ~2 hours"
                     /label=d2EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYKKLSHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV
                     "
     primer_bind     complement(1472..1493)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1733..1752)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2080..2101
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2273..2297)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2375..2394)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2440..2495
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2494..2513)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     enhancer        2850..3229
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3230..3433
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3384..3404
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3460..3469
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3466..4176
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3611..3629)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3879..3898
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(4214..4231)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4220..4444
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4794..4856
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4922..4941)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4923..4941)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4948..4965)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4948..4964)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4957..4979)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5106..5561
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5193..5212)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5403..5424
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5587..5691
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5692..5760,5761..6552)
                     /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(5910..5929)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6723..7311
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7212..7231
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7465..7482
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7599..7620
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7635..7640,7641..7658,7659..7665)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7673..7689
                     /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     7678..7700
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7697..7713
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7697..7713
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        7734..7752
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttaaccctag aaagatagtc tgcgtaaaat tgacgcatgc attcttgaaa tattgctctc
       61 tctttctaaa tagcgcgaat ccgtcgctgt gcatttagga catctcagtc gccgcttgga
      121 gctcccgtga ggcgtgcttg tcaatgcggt aagtgtcact gattttgaac tataacgacc
      181 gcgtgagtca aaatgacgca tgattatctt ttacgtgact tttaagattt aactcatacg
      241 ataattatat tgttatttca tgttctactt acgtgataac ttattatata tatattttct
      301 tgttatagat atcatcaact ttgtatagaa aagttgtccc cctggccact ctgctcaaac
      361 gccccaccct gttctctctg tccccttccc ctgcttaatt tttcttggaa ccacttgtca
      421 ttcctgtcat tgttccacct atgcgtttgg tttcttgttt tctttttttg gggtggagtc
      481 ttgctctgga aagaattttc ttttaattct ccacttgtaa ataacacgtg tcatcagaat
      541 agttaattag gcctgggcat ggtggtgtag tcccagctct ttgggaggcc gaggtcagcg
      601 gattacaagg tcaggtgatc aagaccatcc tggccaacaa acactgtaat tattaaataa
      661 tataattatt tacatataat taaattatac aattattaaa tataattatg tatatgtaat
      721 taaattatat aattattaga taattgttta tatataatta aattattata taataaatta
      781 tataatagat gtttacctgt ccagccccag gtgggctcga cccacaggtc agaggtcatg
      841 agaagctgga tgagaccact ggggatgtcc ctgttttctc agtatatcag ccaaggacct
      901 gaagaacatg ctgtcccagg tcaactaccg ggtccccaac ctcctgacgt caggtgatcc
      961 gcctgcctcg gccccccaaa atgctgggat taaaggcgtg agccacctcg cccagcctct
     1021 ggaattttcc atttaatatt tttggaccaa agttgaccaa ggataactga aaccgaggaa
     1081 atcgaaactg ctgtgaaata gtactaatga agtcaagcaa gtatgacact catactttgt
     1141 tgatggtcat aaaaaataga cagccacatt agataataat atagtaggct tgaccgggcg
     1201 tggtggctca cacctgtaat cccagcactt tgggaggcca agctgctgcc tcgccccgcc
     1261 tcacgccggc gtcctggggg cgcggagagg gcgcccaggt tcaccgcgct ctcagcgcgc
     1321 acttccgggc acggtcaggg cgtgacgtcg ggaggagggc aggtaagtgc cggcgggcgg
     1381 aggagcgggg aggctacaag tttgtacaaa aaagcaggct gccaccatgg tgagcaaggg
     1441 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     1501 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     1561 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     1621 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     1681 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     1741 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     1801 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     1861 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     1921 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     1981 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     2041 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     2101 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagaagctta gccatggctt
     2161 cccgccggag gtggaggagc aggatgatgg cacgctgccc atgtcttgtg cccaggagag
     2221 cgggatggac cgtcaccctg cagcctgtgc ttctgctagg atcaatgtgt agacccagct
     2281 ttcttgtaca aagtggtgat cctcaggtgc aggctgccta tcagaaggtg gtggctggtg
     2341 tggccaatgc cctggctcac aaataccact gagatctttt tccctctgcc aaaaattatg
     2401 gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca
     2461 ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa
     2521 atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgc
     2581 tggctgccat gaacaaaggt tggctataaa gaggtcatca gtatatgaaa cagccccctg
     2641 ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt
     2701 gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca
     2761 gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc ttatggagat
     2821 ccctcgacct gcagcccaag cttcgcgttg acattgatta ttgactagtt attaatagta
     2881 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     2941 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     3001 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     3061 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     3121 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     3181 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     3241 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     3301 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     3361 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     3421 tataagcaga gctctctggc taactagaga acccactgcg ccaccatggt gagcaagggc
     3481 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc
     3541 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     3601 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
     3661 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
     3721 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     3781 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
     3841 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc
     3901 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
     3961 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc
     4021 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac
     4081 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     4141 cactccaccg gcggcatgga cgagctgtac aagtaactcg agtctagagg gcccgtttaa
     4201 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     4261 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     4321 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     4381 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     4441 atggctcgag ttaattaacg agagcataat attgatatgt gccaaagttg tttctgactg
     4501 actaataagt ataatttgtt tctattatgt ataggttaag ctaattactt attttataat
     4561 acaacatgac tgtttttaaa gtacaaaata agtttatttt tgtaaaagag agaatgttta
     4621 aaagttttgt tactttatag aagaaatttt gagtttttgt ttttttttaa taaataaata
     4681 aacataaata aattgtttgt tgaatttatt attagtatgt aagtgtaaat ataataaaac
     4741 ttaatatcta ttcaaattaa taaataaacc tcgatataca gaccgataaa acacatgcgt
     4801 caattttacg catgattatc tttaacgtac gtcacaatat gattatcttt ctagggttaa
     4861 ataatagttt ctaatttttt tattattcag cctgctgtcg tgaataccga gctccaattc
     4921 gccctatagt gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc gtgactggga
     4981 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     5041 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     5101 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     5161 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     5221 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     5281 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     5341 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     5401 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     5461 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     5521 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     5581 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     5641 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     5701 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5761 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5821 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5881 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5941 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     6001 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     6061 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     6121 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     6181 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     6241 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     6301 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     6361 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     6421 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     6481 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     6541 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     6601 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6661 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6721 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6781 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6841 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6901 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6961 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     7021 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     7081 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     7141 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     7201 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     7261 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     7321 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     7381 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     7441 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     7501 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     7561 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     7621 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     7681 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct cgaaattaac
     7741 cctcactaaa gggaacaaaa gctggtacct cgcgcgactt ggtttgccat tctttagcgc
     7801 gcgtcgcgtc acacagcttg gccacaatgt ggtttttgtc aaacgaagat tctatgacgt
     7861 gtttaaagtt taggtcgagt aaagcgcaaa tctttt
//
