LOCUS       sequence-308709-        7791 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  8X copies of the ETS#2 site of the murine Dll4 intronic enhancer and
            a minimal E1b reporter driving expression of EGFP flanked by Tol2 
            sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7791)
  AUTHORS   Fish JE, Flores-Suarez CP, Boudreau E, Herman AM, Gutierrez MC, 
            Gustafson D, DiStefano PV, Cui M, Chen Z, Berman De Ruiz K, 
            Schexnayder TS, Ward CS, Radovanovic I, Wythe JD
  TITLE     Somatic Gain of KRAS Function in the Endothelium is Sufficient to 
            Cause Vascular Malformations that Require MEK but not PI3K 
            Signaling.
  JOURNAL   Circ Res. 2020 Jun 17. doi: 10.1161/CIRCRESAHA.119.316500.
  PUBMED    32552404
REFERENCE   2  (bases 1 to 7791)
  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: "Circ Res. 
            2020 Jun 17. doi: 10.1161/CIRCRESAHA.119.316500."
COMMENT     Sequence Label: JDW 249 (pTol2-Dll4-F2-ETS#2-WT-8X-E1b-EGFP)
FEATURES             Location/Qualifiers
     source          1..7791
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(1232..1243,1244..1406)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    1785..1809
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1935..1959)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     intron          2067..2639
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     regulatory      2713..2722
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2719..2721,2722..2724,2725..3438)
                     /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(2719..2721,2722..2724,2725..3435)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2764..2785)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3025..3044)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3372..3393
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3483..3617
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3507..3526)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3561..3580
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(4054..4241,4242..4253)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4287..4303)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4287..4303)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4340..4360)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4340..4358)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4379..4395)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4379..4395)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4392..4414)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4403..4419
                     /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)."
     promoter        complement(join(4427..4433,4434..4451,4452..4457))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4472..4493
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4610..4627)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4781..5369)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4861..4880)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5540..6331,6332..6400))
                     /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     6163..6182
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6401..6505)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6531..6986)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6668..6689)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6880..6899
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7113..7135
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7127..7144
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7128..7144
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7154..7173
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7154..7172
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 aattatgatt aacaagtaca tgttaaataa agattttaat atgaatgcca ccactggagc
       61 attcatgcca tttggagctt cttcctgttt ggatcactag aaggaggagg tcactcatta
      121 cagttctcat atacagtcgt tggttggttg gttggttggt agattgattg attgattgat
      181 tgattgattg attgattgat tgattgattg attgattgat tgattgattg attgattggt
      241 agtcaaaata agaaataatt tccacagatt cattacagaa atgattaaat gcatacataa
      301 aaaactgggg ggggggggga tacaacaaca cacttaagtc acatttgcct acgtaaagaa
      361 aagtaaagaa aatcaatagc tatattttac atctcctttt tttgctgtct tttaattagc
      421 cttgttttgc tgttatcttg attgaaactg taacttcttc acctgcttct tttctttgta
      481 ggttttgcca gcccagaaac aggcatggct gtgcaaggcc agagcaccat gcacaatgcg
      541 ctgcatgtct tcatgaacgg ctcaatgtcc tcagtccagg gctcagccaa cgaccccatc
      601 ttcctccttc accatgcttt cattgacagg taacaaacac gtcatgacat tagactgcac
      661 agtttttgac aaagttcata caatctgttg tttatagctg ctacaattag tgaagtttgt
      721 gaatgtactt ggatgagcag cgaaagatca attgagatca attgttagag tttggttgcc
      781 ctgcagagca aagaacaaaa aataatctgg tggctttact gcgtgaggtt attattggtg
      841 gaatagaaac acaaaacata attgcattta tttgtttaat tttttatctt atcttaactt
      901 tcatcttgca tatttgtttc ttacatcatt tctagcatct ttgagcgctg gctaagaact
      961 catcagcctc cccggtccat ctacccacgt accaatgcac caattggcca caatgacggc
     1021 tactacatgg tgccattcct tcctctttat aggaatggag actacctcct gtccaacaat
     1081 gctcttggat acgagtacgc ctacctgttg gacccaggtc attgcacaac accagaaatg
     1141 ccctctgatc tgcaaaagac gtgaatatct gttcagacac ccatatccac tctgttccac
     1201 acaggtcaga ggtttgtcca ggagttcttg acagaggtgt aaaaagtact caaaaatttt
     1261 actcaagtga aagtacaagt acttagggaa aattttactc aattaaaagt aaaagtatct
     1321 ggctagaatc ttacttgagt aaaagtaaaa aagtactcca ttaaaattgt acttgagtat
     1381 taaggaagta aaagtaaaag caagaaagaa aactagagat tcttgtttaa gcttttaatc
     1441 tcaaaaaaca ttaaatgaaa tgcatacaag gttttatcct gctttagaac tgtttgtatt
     1501 taattatcaa actataagac agacaatcta atgccagtac acgctactca aagttgtaaa
     1561 acctcagatt taacttcagt agaagctgat tctcaaaatt gttagtgtca agcctagctc
     1621 ttttggggct gaaaagcaat cctgcagtgc tgaaaagcct ctcacaggca gccgatgcgg
     1681 gaagaggtgt attagtcttg atagagaggc tgcaaatagc aggaaacgtg agcagagact
     1741 ccctggtgtc tgaaacacag gccagatggg ccctcgagga tatcacaagt ttgtacaaaa
     1801 aagcaggctg gatccgcgtt tcctgcgggg cgtttcctgc ggggcgtttc ctgcggggcg
     1861 tttcctgcgg ggcgtttcct gcggggcgtt tcctgcgggg cgtttcctgc ggggcgtttc
     1921 ctgcggggcg gccgccccag ctttcttgta caaagtggtg atatcaagat ctctcgactc
     1981 tagagggtat ataatggatc ccatcgcgtc tcagcctcac tttgagctcc tccacacgaa
     2041 tttcgaccga tcctgagaac ttcagggtga gtttggggac ccttgattgt tctttctttt
     2101 tcgctattgt aaaattcatg ttatatggag ggggcaaagt tttcagggtg ttgtttagaa
     2161 tgggaagatg tcccttgtat caccatggac cctcatgata attttgtttc tttcactttc
     2221 tactctgttg acaaccattg tctcctctta ttttcttttc attttctgta actttttcgt
     2281 taaactttag cttgcatttg taacgaattt ttaaattcac ttttgtttat ttgtcagatt
     2341 gtaagtactt tctctaatca cttttttttc aaggcaatca gggtatatta tattgtactt
     2401 cagcacagtt ttagagaaca attgttataa ttaaatgata aggtagaata tttctgcata
     2461 taaattctgg ctggcgtgga aatattctta ttggtagaaa caactacacc ctggtcatca
     2521 tcctgccttt ctctttatgg ttacaatgat atacactgtt tgagatgagg ataaaatact
     2581 ctgagtccaa accgggcccc tctgctaacc atgttcatgc cttcttctct ttcctacagc
     2641 tcctgggcaa cgtgctggtt gttgtgctgt ctcatcattt tggcaaagaa ttcctcgacg
     2701 gatccaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     2761 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     2821 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     2881 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     2941 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     3001 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     3061 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     3121 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     3181 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     3241 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     3301 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     3361 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     3421 gacgagctgt acaagtaaag cggccgccac cgcggtggag ctcgaattaa ttcatcgatg
     3481 atgatccaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt
     3541 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
     3601 gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg
     3661 aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg gctgattatg
     3721 atcctctaga tcagatccga agatacggcc acgggtgctc ttgatcctgt ggctgatttt
     3781 ggactgtgct gctcgcagct gctgatgaat cacatacttc ctccattttc ttccactgat
     3841 tgactgttat aatttcccta atttccaggt caaggtgctg tgcattgtgg taatagatgt
     3901 gacatgacgt cacttccaaa ggaccaatga acatgtctga ccaatttcat ataatgtgaa
     3961 aacgattttc ataggcagaa taaataacat ttaaattaaa ctgggcatca gcgcaattca
     4021 attggtttgg taatagcaag ggaaaataga atgaagtgat ctccaaaaaa taagtacttt
     4081 ttgactgtaa ataaaattgt aaggagtaaa aagtactttt ttttctaaaa aaatgtaatt
     4141 aagtaaaagt aaaagtattg atttttaatt gtactcaagt aaagtaaaaa tccccaaaaa
     4201 taatacttaa gtacagtaat caagtaaaat tactcaagta ctttacacct ctggttcttg
     4261 accccctacc ttcagcaagc ccagcagatc cactagttct agagcggccg ccaccgcggt
     4321 ggagctccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt
     4381 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4441 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4501 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4561 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4621 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4681 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4741 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4801 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4861 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4921 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4981 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5041 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5101 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5161 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5221 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5281 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5341 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5401 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5461 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5521 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5581 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5641 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5701 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5761 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5821 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5881 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5941 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6001 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6061 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6121 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6181 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6241 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6301 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6361 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6421 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6481 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag
     6541 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     6601 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     6661 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     6721 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     6781 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     6841 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     6901 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     6961 gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga
     7021 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
     7081 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc
     7141 cagtgagcgc gcgtaatacg actcactata gggcgaattg ggtaccaaat agtaggaatt
     7201 acccacctgt acaagtgctg aaaacttgga tgaataagcc cgtttgccat tttctactgc
     7261 tattttaaat cttttctgta tttgtctgca tttgtcttta cccttgaata aatgttttag
     7321 ttgttttttt tcaattatga ctgtgtttaa cagacattat tacaataata tgtaatagta
     7381 gtacacacta ttattatgta atagtacttg ttgactgtat ttgagaactg gaccgagtga
     7441 gtgttacgtc acccattcaa aatgacttac ttctggctcc aacaaaatga agttaattca
     7501 gttgccattt ttcactgtat ggacatcgcc gtgttggagc tagacgttgc caataagcaa
     7561 gaaagagccg agatgcgtcg agtctgagtc accgttccta tggcaacccc tctaaccaat
     7621 cagaagtaag cttgttggaa gtccacagcc taccacttga aagcgggctg cacaaaatct
     7681 gtcaaacgtt ttgaacgttg gatgtgagag cacatacttt tattaaggca tctggttggt
     7741 cagtttataa tatcaacaac ttgggctaca gaaaagaaaa gttattacag a
//
