LOCUS       sequence-453875-        6220 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes destablized mCherry under IBRE4 promoter, and constitutive 
            BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6220)
  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 6220)
  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: pHM131
FEATURES             Location/Qualifiers
     source          1..6220
                     /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             1188..1895
                     /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(1333..1351)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1601..1620
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2068..2189
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2079..2098)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2133..2152
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2306..2326)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2311..2640
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2491..2626
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2650..3348
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     repeat_region   3625..3687
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3799..3818)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3918..3940
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3978..3996)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4064..4168
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4169..4237,4238..5029)
                     /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(4387..4406)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5200..5788
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5689..5708
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5942..5959
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6076..6097
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6112..6117,6118..6135,6136..6142)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6150..6166
                     /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     6155..6177
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6174..6190
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6174..6190
                     /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 gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg
     1261 gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     1321 gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca
     1381 tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc
     1441 ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     1501 aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct
     1561 acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga
     1621 ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg
     1681 agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca
     1741 cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg
     1801 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc
     1861 gccactccac cggcggcatg gacgagctgt acaagaagct tagccatggc ttcccgccgg
     1921 aggtggagga gcaggatgat ggcacgctgc ccatgtcttg tgcccaggag agcgggatgg
     1981 accgtcaccc tgcagcctgt gcttctgcta ggatcaatgt gtgataattc tagagtcggg
     2041 gcggccggcc gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa
     2101 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     2161 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     2221 aggttcaggg ggaggtgtgg gaggcctcga ccaaggcagc tgtagatctt agccatctgc
     2281 tttttgcttg tactgtcgac cctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     2341 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     2401 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     2461 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     2521 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     2581 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     2641 aagcttacca tgagcgagct gattaaggag aacatgcaca tgaagctgta catggagggc
     2701 accgtggaca accatcactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc
     2761 acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc
     2821 ctggctacta gcttcctcta cggcagcaag accttcatca accacaccca gggcatcccc
     2881 gacttcttca agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa
     2941 gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac
     3001 aacgtcaaga tcagaggggt gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca
     3061 ctcggctggg aggccttcac cgagacgctg taccccgctg acggcggcct ggaaggcaga
     3121 aacgacatgg ccctgaagct cgtgggcggg agccatctga tcgcaaacat caagaccaca
     3181 tatagatcca agaaacccgc taagaacctc aagatgcctg gcgtctacta tgtggactac
     3241 agactggaaa gaatcaagga ggccaacaac gagacctacg tcgagcagca cgaggtggca
     3301 gtggccagat actgcgacct ccctagcaaa ctggggcaca agcttaatgc ggccgcaact
     3361 cccacctgca acatgcgtga ctaacgcccg ccccacgacc cgcagcgccc gaccgaaagg
     3421 agcgcacgac cccatgcatc gatactagtt aaaagttttg ttactttata gaagaaattt
     3481 tgagtttttg ttttttttta ataaataaat aaacataaat aaattgtttg ttgaatttat
     3541 tattagtatg taagtgtaaa tataataaaa cttaatatct attcaaatta ataaataaac
     3601 ctcgatatac agaccgataa aacacatgcg tcaattttac gcatgattat ctttaacgta
     3661 cgtcacaata tgattatctt tctagggtta atctagtata cgcgtatgcg gccgcttaat
     3721 taatccatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3781 catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     3841 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     3901 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     3961 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     4021 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     4081 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     4141 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     4201 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     4261 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     4321 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     4381 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     4441 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     4501 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     4561 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     4621 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     4681 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     4741 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     4801 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     4861 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     4921 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     4981 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     5041 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     5101 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     5161 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5221 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5281 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5341 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     5401 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5461 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5521 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5581 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5641 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     5701 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5761 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5821 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     5881 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     5941 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     6001 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     6061 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     6121 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     6181 cagctatgac catgattacg ccggggcgcg ccattctaga
//
