LOCUS       sequence-453800-        5883 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes constitutive GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5883)
  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 5883)
  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: pHM103
FEATURES             Location/Qualifiers
     source          1..5883
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   23..57
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    616..634
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    637..655
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     intron          700..1638
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1595..1615
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1680..1689
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1686..1688,1689..1691,1692..2405)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1686..1688,1689..1691,1692..2402)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1731..1752)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1992..2011)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2339..2360
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2506..3094
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2559..2579)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2977..2988)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   3306..3368
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3480..3499)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3599..3621
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3659..3677)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3745..3849
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3850..3918,3919..4710)
                     /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(4068..4087)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4881..5469
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5370..5389
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5623..5640
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5757..5778
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5793..5798,5799..5816,5817..5823)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5831..5847
                     /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     5836..5858
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5855..5871
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5855..5871
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ggggcgcgcc attctagatt aaccctagaa agatagtctg cgtaaaattg acgcatgcat
       61 tcttgaaata ttgctctctc tttctaaata gcgcgaatcc gtcgctgtgc atttaggaca
      121 tctcagtcgc cgcttggagc tcccgtgagg cgtgcttgtc aatgcggtaa gtgtcactga
      181 ttttgaacta taacgaccgc gtgagtcaaa atgacgcatg attatctttt acgtgacttt
      241 taagatttaa ctcatacgat aattatattg ttatttcatg ttctacttac gtgataactt
      301 attatatata tattttcttg ttatagatat caactagaat gctagcatgg gcccagatct
      361 ccgaggggac ccgacaggcc cgcagggaca gcagagatcc agtttggatc gataagcttg
      421 atatcgaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga
      481 gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa
      541 ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta
      601 tataagtgca ggatctccct atcagtgata gagatctccc tatcagtgat agagatcgtc
      661 gacgttcttt ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt gtgtggttcc
      721 cgcgggcctg gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccacctgg
      781 ctgcagtacg tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg
      841 ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg
      901 ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct
      961 ctagccattt aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt
     1021 gtaaatgcgg gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac
     1081 ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg
     1141 agaatcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg
     1201 ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg
     1261 gaaagatggc cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg
     1321 ggagagcggg cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc
     1381 gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc
     1441 ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac
     1501 actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa
     1561 tttgcccttt ttaagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt
     1621 ttttttcttc catttcaggt gtcgtgaggg gatctggagc tctcgagaat tctcacgcgg
     1681 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1741 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1801 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1861 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     1921 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1981 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     2041 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     2101 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     2161 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     2221 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     2281 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     2341 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     2401 agtaacagat cggcgtgatg gaagagtaaa gcggccgcga ctctagagtc gacctgcagg
     2461 catgcaagct tgcaggcatg caagcttgat atcaagctta tcgataatca acctctggat
     2521 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     2581 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     2641 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     2701 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     2761 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     2821 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     2881 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
     2941 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     3001 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     3061 acgagtcgga tctccctttg ggccgcctcc ccgccgaaag gagcgcacga ccccatgcat
     3121 cgatactagt taaaagtttt gttactttat agaagaaatt ttgagttttt gttttttttt
     3181 aataaataaa taaacataaa taaattgttt gttgaattta ttattagtat gtaagtgtaa
     3241 atataataaa acttaatatc tattcaaatt aataaataaa cctcgatata cagaccgata
     3301 aaacacatgc gtcaatttta cgcatgatta tctttaacgt acgtcacaat atgattatct
     3361 ttctagggtt aatctagtat acgcgtatgc ggccgcttaa ttaatccatg gcgcctgatg
     3421 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt
     3481 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     3541 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     3601 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     3661 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     3721 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     3781 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     3841 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     3901 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     3961 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     4021 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     4081 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     4141 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     4201 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     4261 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     4321 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     4381 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     4441 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     4501 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     4561 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     4621 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     4681 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     4741 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     4801 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     4861 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4921 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4981 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     5041 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     5101 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     5161 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     5221 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     5281 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     5341 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     5401 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     5461 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     5521 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     5581 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     5641 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     5701 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     5761 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     5821 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     5881 gcc
//
