LOCUS       sequence-453798-        6229 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes destablized GFP under IBRE4 promoter, and constitutive BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6229)
  AUTHORS   McNamara HM, Solley SC, Adamson B, Chan MM, Toettcher JE
  TITLE     Recording morphogen signals reveals mechanisms underlying gastruloid
            symmetry breaking.
  JOURNAL   Nat Cell Biol. 2024 Oct 2. doi: 10.1038/s41556-024-01521-9.
  PUBMED    39358450
REFERENCE   2  (bases 1 to 6229)
  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 Cell 
            Biol. 2024 Oct 2. doi: 10.1038/s41556-024-01521-9."
COMMENT     Sequence Label: pHM130
FEATURES             Location/Qualifiers
     source          1..6229
                     /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)"
     polyA_signal    368..416
                     /note="synthetic polyadenylation signal"
     misc_feature    430..521
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     470..489
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        1095..1126
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     regulatory      1182..1191
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1188..1190,1191..1193,1194..1904,1905..1910,
                     1911..2033)
                     /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(1233..1254)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1494..1513)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1841..1862
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2077..2198
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2088..2107)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2142..2161
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2315..2335)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2320..2649
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2500..2635
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2659..3357
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     repeat_region   3634..3696
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3808..3827)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3927..3949
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3987..4005)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4073..4177
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4178..4246,4247..5038)
                     /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(4396..4415)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5209..5797
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5698..5717
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5951..5968
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6085..6106
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6121..6126,6127..6144,6145..6151)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6159..6175
                     /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     6164..6186
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6183..6199
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6183..6199
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
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 atcaactaga atgctagcat gggcccagat ctccgaggag gtacttggag
      361 cggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt tgtgtgaatc
      421 gatagtacta acatacgctc tccatcaaaa caaaacgaaa caaaacaaac tagcaaaata
      481 ggctgtcccc agtgcaagtg caggtgccag aacatttctc tatcgatagg taccgagctc
      541 ggccgcagac gtggaggagc agagcgccga ggctggcgcc aggcggcctc ttttgtttat
      601 ctgacagcta aatatcaagg caatcaccgc ctcgcggccc tgcctccaac ccccacagct
      661 aagacaaaca gacgcgtggc cgcagacgtg gaggagcaga gcgccgaggc tggcgccagg
      721 cggcctcttt tgtttatctg acagctaaat atcaaggcaa tcaccgcctc gcggccctgc
      781 ctccaacccc cacagctaag acaaacagcc cgggggccgc agacgtggag gagcagagcg
      841 ccgaggctgg cgccaggcgg cctcttttgt ttatctgaca gctaaatatc aaggcaatca
      901 ccgcctcgcg gccctgcctc caacccccac agctaagaca aacagacgcg tggccgcaga
      961 cgtggaggag cagagcgccg aggctggcgc caggcggcct cttttgttta tctgacagct
     1021 aaatatcaag gcaatcaccg cctcgcggcc ctgcctccaa cccccacagc taagacaaac
     1081 agagatctag actctagagg gtatataatg gaagctcgaa ttccagcttg gcattccggt
     1141 actgttggta aaaagcttgg cattccggta ctgttggtaa agccaccatg gtgagcaagg
     1201 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     1261 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     1321 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     1381 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     1441 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     1501 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1561 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1621 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     1681 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     1741 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     1801 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1861 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagaagctt agccatggct
     1921 tcccgccgga ggtggaggag caggatgatg gcacgctgcc catgtcttgt gcccaggaga
     1981 gcgggatgga ccgtcaccct gcagcctgtg cttctgctag gatcaatgtg tgataattct
     2041 agagtcgggg cggccggccg cttcgagcag acatgataag atacattgat gagtttggac
     2101 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     2161 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     2221 ttatgtttca ggttcagggg gaggtgtggg aggcctcgac caaggcagct gtagatctta
     2281 gccatctgct ttttgcttgt actgtcgacc ctgtggaatg tgtgtcagtt agggtgtgga
     2341 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2401 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2461 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     2521 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     2581 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     2641 ttttgcaaaa agcttaccat gagcgagctg attaaggaga acatgcacat gaagctgtac
     2701 atggagggca ccgtggacaa ccatcacttc aagtgcacat ccgagggcga aggcaagccc
     2761 tacgagggca cccagaccat gagaatcaag gtggtcgagg gcggccctct ccccttcgcc
     2821 ttcgacatcc tggctactag cttcctctac ggcagcaaga ccttcatcaa ccacacccag
     2881 ggcatccccg acttcttcaa gcagtccttc cctgagggct tcacatggga gagagtcacc
     2941 acatacgaag acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc
     3001 ctcatctaca acgtcaagat cagaggggtg aacttcacat ccaacggccc tgtgatgcag
     3061 aagaaaacac tcggctggga ggccttcacc gagacgctgt accccgctga cggcggcctg
     3121 gaaggcagaa acgacatggc cctgaagctc gtgggcggga gccatctgat cgcaaacatc
     3181 aagaccacat atagatccaa gaaacccgct aagaacctca agatgcctgg cgtctactat
     3241 gtggactaca gactggaaag aatcaaggag gccaacaacg agacctacgt cgagcagcac
     3301 gaggtggcag tggccagata ctgcgacctc cctagcaaac tggggcacaa gcttaatgcg
     3361 gccgcaactc ccacctgcaa catgcgtgac taacgcccgc cccacgaccc gcagcgcccg
     3421 accgaaagga gcgcacgacc ccatgcatcg atactagtta aaagttttgt tactttatag
     3481 aagaaatttt gagtttttgt ttttttttaa taaataaata aacataaata aattgtttgt
     3541 tgaatttatt attagtatgt aagtgtaaat ataataaaac ttaatatcta ttcaaattaa
     3601 taaataaacc tcgatataca gaccgataaa acacatgcgt caattttacg catgattatc
     3661 tttaacgtac gtcacaatat gattatcttt ctagggttaa tctagtatac gcgtatgcgg
     3721 ccgcttaatt aatccatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3781 ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3841 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     3901 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     3961 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     4021 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     4081 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4141 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     4201 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     4261 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     4321 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     4381 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     4441 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     4501 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     4561 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     4621 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     4681 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     4741 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     4801 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     4861 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     4921 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     4981 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     5041 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     5101 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     5161 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     5221 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     5281 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     5341 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     5401 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     5461 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     5521 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     5581 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     5641 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     5701 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     5761 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     5821 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     5881 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     5941 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     6001 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     6061 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
     6121 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
     6181 cacaggaaac agctatgacc atgattacgc cggggcgcgc cattctaga
//
