LOCUS       sequence-421747-        7666 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 7666)
  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 7666)
  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-MGT1-d2EGFP_CMV-mCherry
FEATURES             Location/Qualifiers
     source          1..7666
                     /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    1166..1190
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1191..1200
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1197..1199,1200..1202,1203..1913,1914..1919,
                     1920..2042)
                     /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(1242..1263)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1503..1522)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1850..1871
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2043..2067)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2145..2164)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2210..2265
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2264..2283)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     enhancer        2620..2999
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3000..3203
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3154..3174
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3230..3239
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3236..3946
                     /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(3381..3399)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3649..3668
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3984..4001)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3990..4214
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4564..4626
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4692..4711)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4693..4711)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4718..4735)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4718..4734)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4727..4749)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4876..5331
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4963..4982)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5173..5194
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5357..5461
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5462..5530,5531..6322)
                     /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(5680..5699)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6493..7081
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6982..7001
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7235..7252
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7369..7390
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7405..7410,7411..7428,7429..7435)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7443..7459
                     /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     7448..7470
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7467..7483
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7467..7483
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        7504..7522
                     /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 aagttgatat ttatttttag gaccagaaag
      361 ttaaagtgaa ttggatttga tccattttct gaaaggctgg caagaattct tgacattgca
      421 caggaatttc catgtcagca tgttctcaca tgtatgatct aatttaggtg acacttcctg
      481 gctcaggaag ttgaatttca ttaagccttt gtggtttggg gctctgctgt gctttgacag
      541 ctctgatctc ctcccttccg gctgggctgt ctggggcgct ctaaaatgag tgttgattta
      601 atgcactgcc ttcgcacccg tgctggtaga ttattttggg gggcgggggt tgaggaaatg
      661 gcatgactca gagtttaaaa gccccaaatc ttagctgtgc ctgtgtagct ttaccacata
      721 acccattgat aacttagttg tgcaaccatc accaccatct gttttcagaa ctcttttcat
      781 tttgcgaaac tgaaacccgt taagcactga tttcccactc tccctcctcc cagcccatag
      841 caaaccacca tcccaccagc actttcattt cgcaaatggc aaaactgaag ccgatattgt
      901 ggttgtgact tatcccaaag taatatacac ataaacctct atggatgagg aaaaagacag
      961 agggaaacta aaaattcaaa agaacaaatt tgactcacag atttgctgac tcatagtttc
     1021 gctttcgctt ttgctttggc ccccgagggc tcggagaggt gcaaagcggt gacagatccg
     1081 tgactcagcg tccccacccc tgctggggaa ggagccccgc cggccgccgc ggcaccgcac
     1141 agccacccct gtccccggga cgcctacaag tttgtacaaa aaagcaggct gccaccatgg
     1201 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     1261 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     1321 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     1381 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     1441 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     1501 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     1561 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     1621 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     1681 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     1741 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     1801 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     1861 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagaagctta
     1921 gccatggctt cccgccggag gtggaggagc aggatgatgg cacgctgccc atgtcttgtg
     1981 cccaggagag cgggatggac cgtcaccctg cagcctgtgc ttctgctagg atcaatgtgt
     2041 agacccagct ttcttgtaca aagtggtgat cctcaggtgc aggctgccta tcagaaggtg
     2101 gtggctggtg tggccaatgc cctggctcac aaataccact gagatctttt tccctctgcc
     2161 aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa
     2221 tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat
     2281 gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat
     2341 gcccatatgc tggctgccat gaacaaaggt tggctataaa gaggtcatca gtatatgaaa
     2401 cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt
     2461 tttatatttt gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt
     2521 ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc
     2581 ttatggagat ccctcgacct gcagcccaag cttcgcgttg acattgatta ttgactagtt
     2641 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     2701 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     2761 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     2821 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     2881 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     2941 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     3001 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
     3061 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     3121 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
     3181 gggaggtcta tataagcaga gctctctggc taactagaga acccactgcg ccaccatggt
     3241 gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca
     3301 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
     3361 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc
     3421 ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc
     3481 cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat
     3541 gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga
     3601 gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca
     3661 gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct
     3721 gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt
     3781 caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat
     3841 caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc
     3901 cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtaactcg agtctagagg
     3961 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     4021 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     4081 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     4141 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     4201 ggtgggctct atggctcgag ttaattaacg agagcataat attgatatgt gccaaagttg
     4261 tttctgactg actaataagt ataatttgtt tctattatgt ataggttaag ctaattactt
     4321 attttataat acaacatgac tgtttttaaa gtacaaaata agtttatttt tgtaaaagag
     4381 agaatgttta aaagttttgt tactttatag aagaaatttt gagtttttgt ttttttttaa
     4441 taaataaata aacataaata aattgtttgt tgaatttatt attagtatgt aagtgtaaat
     4501 ataataaaac ttaatatcta ttcaaattaa taaataaacc tcgatataca gaccgataaa
     4561 acacatgcgt caattttacg catgattatc tttaacgtac gtcacaatat gattatcttt
     4621 ctagggttaa ataatagttt ctaatttttt tattattcag cctgctgtcg tgaataccga
     4681 gctccaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc
     4741 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     4801 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     4861 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4921 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     4981 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     5041 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     5101 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     5161 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     5221 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     5281 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     5341 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5401 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5461 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5521 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5581 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5641 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5701 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5761 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5821 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5881 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5941 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     6001 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     6061 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     6121 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     6181 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     6241 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     6301 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     6361 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6421 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6481 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6541 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6601 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6661 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6721 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6781 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6841 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6901 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6961 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     7021 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     7081 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     7141 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     7201 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     7261 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     7321 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     7381 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     7441 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct
     7501 cgaaattaac cctcactaaa gggaacaaaa gctggtacct cgcgcgactt ggtttgccat
     7561 tctttagcgc gcgtcgcgtc acacagcttg gccacaatgt ggtttttgtc aaacgaagat
     7621 tctatgacgt gtttaaagtt taggtcgagt aaagcgcaaa tctttt
//
