LOCUS       sequence-178754-        7950 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  contains dll4 enhancer 2 sequence, and dll4 specific gene promoter 
            driving expression of eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7950)
  AUTHORS   Quillien A, Abdalla M, Yu J, Ou J, Zhu LJ, Lawson ND
  TITLE     Robust Identification of Developmentally Active Endothelial 
            Enhancers in Zebrafish Using FANS-Assisted ATAC-Seq.
  JOURNAL   Cell Rep. 2017 Jul 18;20(3):709-720. doi: 
            10.1016/j.celrep.2017.06.070.
  PUBMED    28723572
REFERENCE   2  (bases 1 to 7950)
  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"; doi: 
            "10.1016/j.celrep.2017.06"; journalName: "Cell Rep"; date: 
            "2017-07-18- 18"; volume: "20"; issue: "3"; pages: "709-720"
COMMENT     Sequence Label: pT2-cryR;dll4CE2-P1Egfp
FEATURES             Location/Qualifiers
     source          1..7950
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     643..659
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    679..699
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    1292..1316
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1972..1981
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1978..1980,1981..1983,1984..2694)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1978..1980,1981..1983,1984..2694)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2023..2044)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2284..2303)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2631..2652
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2695..2760
                     /label=MCS
                     /note="multiple cloning site"
     protein_bind    complement(2781..2805)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2831..2851)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(2859..2875)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2891..3025
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(3464..3651,3652..3663)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3697..3713)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3697..3713)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3750..3770)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3750..3768)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3789..3805)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3802..3824)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3813..3829
                     /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(3837..3843,3844..3861,3862..3867))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3882..3903
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4020..4037)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4191..4779)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4271..4290)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4950..5741,5742..5810))
                     /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     5573..5592
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5811..5915)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6078..6099)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(6448..6459,6460..6622)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     polyA_signal    6991..7112
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(7235..7945)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEDDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGHHSTGGMDELYK"
     primer_bind     complement(7315..7338)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(7513..7532)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     7782..7800
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      join(7942..7950,1)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 cgaccggtaa tgtcagacct ggtaactcct tactgtaact tattgcacct gatgaacaaa
       61 agatgtgaac tgaacgcaag taggaggcta atagaggcta gtacagtaca aagatcccca
      121 aatgatgtgt gccggcctta gtggaggcag acccctggtt atatacctgt gctgcagaaa
      181 gaggggctta caaatggatc tctctggaat ggcattatct catggtggga acagtgttgt
      241 cagcagaatc agcggggtgg actctgacct gagaatgaca gtctagcact gaatggctca
      301 gacaatactc ttgtggccaa ctatcaccgg aaaataagtg cctgccaaaa caaaccagac
      361 cagatgaagc tacatggctg tagaacgtca gtatttaatg tgttgtggcc acttggttca
      421 ccagatttaa gagctaattt ttttaaatat cacagataat caatcgcttt tttttttctt
      481 gactgaaaag cttagactgg aaaattcttt gaaaaagttt gcactacaga aaataaatac
      541 ataacatcct gcactgaatg cacactatta atagaaaaga gagcattatt gtaggaaatg
      601 tattgtatat gtacattctg tgcatttcac aattttctcg agcaggaaac agctatgacc
      661 atgattacgc caagctatca actttgtata gaaaagttgt tgttgtacat ttacatgtca
      721 aaaatttggc tttccattca tgagaaacac ataagtcagg tccaaaaacc cgccagtttg
      781 ctgtctggtg agaaaccagg aagtaacaaa gcactgaaga tcaatgagag cgaggcggtg
      841 acaggaaggg gaggagctag agccgtccaa tgacaggcag gcctcaggga gcagcaactg
      901 acgcaaacca ggaagccacc tgatgtgact ggggtcagat aaacgcaggc cgcggaagaa
      961 caggtgcttc ctctgcgctc gctgccttct tcggccatgt cagactggtg tgtgtgtgtg
     1021 tgtatttgtg tgtgtgcaca tgagtgtgta tgtgcgtgtg tggagcgttt aaaggaacag
     1081 catcctcggg cgggtcgaga cgaaggacag tgcacgcgca caggccagga aacgccgcac
     1141 aactccggct gtttttgttt ttgttcagcc ggtctgtttc tatatacatg ctgtaaacac
     1201 acatttactc ttcgaaaaaa gaagaagtcg ttcagaatta ctcttcaagg actcaacctc
     1261 atctgctcct aatataggca cttgtcaaaa agcaagtttg tacaaaaaag caggctcgct
     1321 aggatactaa aatgcgcttt caggctgtcc cagcttgaaa catgaagagg ctctgctccg
     1381 gaacatcagg taaggcatga ccctccactg accgtgcacg ctttcagtga tgtaatataa
     1441 ttatccgcgc aattaactgg aacgaggaca aacgacaagt aataggctgt gcgttgtgcg
     1501 aagtttcatc cgatttagtt tttcttcaga tacgcgtatt ctgcactgca tggtgtgaaa
     1561 tggtcagtat ttaatcgtgc ctgctttaaa tatgccaaca gatgctgggc ttgttaataa
     1621 tcactcgcgc tcaattggtc atcatcatcc gcgtagtggt gggtattaaa tgtggatatc
     1681 attgttgcca agagaggggg gctgactgag cataagtggg cggcgtttag cgatagacaa
     1741 gagtttatat aatgagcagc gcggacacaa agccagtata aatactgagg gacgtaagaa
     1801 acaagtcttc attcacgaat tacacgagaa gcgcgaggat gcccaacagc gcaatgctga
     1861 cgcacaaagt ttagaagcaa aacaagacat aaccattgac actaaacaca tcggtgcgct
     1921 cactgtggaa taccaaacat gctcagtgga tatcttgaga cgaagaggat cgccaccatg
     1981 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     2041 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     2101 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     2161 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     2221 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     2281 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     2341 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     2401 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     2461 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     2521 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2581 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     2641 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtccgga
     2701 ctcagatctc gagctcaagc ttcgaattct gcagtcgacg gtaccgcggg cccgggatcc
     2761 accggatcta gataactgat acccagcttt cttgtacaaa gtggggcgcg cctacgtaac
     2821 tagtctcgag caactttatt atacatagtt gataattcac tggccgtcgt tttacggtac
     2881 catcgatgat gatccagaca tgataagata cattgatgag tttggacaaa ccacaactag
     2941 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     3001 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     3061 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtatggc
     3121 tgattatgat cctctagatc agatctgcga agatacggcc acgggtgctc ttgatcctgt
     3181 ggctgatttt ggactgtgct gctcgcagct gctgatgaat cacatacttc ctccattttc
     3241 ttccactgat tgactgttat aatttcccta atttccaggt caaggtgctg tgcattgtgg
     3301 taatagatgt gacatgacgt cacttccaaa ggaccaatga acatgtctga ccaatttcat
     3361 ataatgtgaa aacgattttc ataggcagaa taaataacat ttaaattaaa ctgggcatca
     3421 gcgcaattca attggtttgg taatagcaag ggaaaataga atgaagtgat ctccaaaaaa
     3481 taagtacttt ttgactgtaa ataaaattgt aaggagtaaa aagtactttt ttttctaaaa
     3541 aaatgtaatt aagtaaaagt aaaagtattg atttttaatt gtactcaagt aaagtaaaaa
     3601 tccccaaaaa taatacttaa gtacagtaat caagtaaaat tactcaagta ctttacacct
     3661 ctggttcttg accccctacc ttcagcaagc ccagcagatc cactagttct agagcggccg
     3721 ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg
     3781 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     3841 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     3901 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3961 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     4021 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4081 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4141 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4201 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4261 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4321 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4381 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4441 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4501 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4561 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4621 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4681 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4741 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4801 acggggtctg acgctcagtg gaacgaaaac tcacgttaag gcattttggt catgagatta
     4861 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4921 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4981 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     5041 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     5101 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     5161 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     5221 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     5281 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     5341 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5401 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5461 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5521 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5581 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5641 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5701 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5761 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     5821 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     5881 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5941 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     6001 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     6061 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     6121 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc acgctggcta agaactcatc
     6181 agcctccccg gtccatctac ccacgtacca atgcaccaat tggccacaat gacggctact
     6241 acatggtgcc attccttcct ctttatagga atggagacta cctcctgtcc aacaatgctc
     6301 ttggatacga gtacgcctac ctgttggacc caggtcattg cacaacacca gaaatgccct
     6361 ctgatctgca aaagacgtga atatctgttc agacacccat atccactctg ttccacacag
     6421 gtcagaggtt tgtccaggag ttcttgacag aggtgtaaaa agtactcaaa aattttactc
     6481 aagtgaaagt acaagtactt agggaaaatt ttactcaatt aaaagtaaaa gtatctggct
     6541 agaatcttac ttgagtaaaa gtaaaaaagt actccattaa aattgtactt gagtattaag
     6601 gaagtaaaag taaaagcaag aaagaaaact agagattctt gtttaagctt ttaatctcaa
     6661 aaaacattaa atgaaatgca tacaaggttt tatcctgctt tagaactgtt tgtatttaat
     6721 tatcaaacta taagacagac aatctaatgc cagtacacgc tactcaaagt tgtaaaacct
     6781 cagatttaac ttcagtagaa gctgattctc aaaattgtta gtgtcaagcc tagctctttt
     6841 ggggctgaaa agcaatcctg cagtgctgaa aagcctctca caggcagccg atgcgggaag
     6901 aggtgtatta gtcttgatag agaggctgca aatagcagga aacgtgagca gagactccct
     6961 ggtgtctgaa acacaggccc atttacgcct taagatacat tgatgagttt ggacaaacca
     7021 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     7081 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     7141 ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
     7201 gtatggctga ttatgatcta gagtcgcggc cgctttactt gtacagctcg tccatgccgc
     7261 cggtggagtg gtggccctcg gcgcgctcgt actgttccac gatggtgtag tcctcgttgt
     7321 gggaggtgat gtccagcttg atgttgacgt tgtaggcgcc gggcagctgc acgggcttct
     7381 tggccttgta ggtggtcttg acctcggcgt cgtagtggcc gccgtccttc agcttcagcc
     7441 tctgcttgat ctcgcccttc agggcgccgt cctcggggta catccgctcg gaggaggcct
     7501 cccagcccat ggtcttcttc tgcattacgg ggccgtcgga ggggaagttg gtgccgcgca
     7561 gcttcacctt gtagatgaac tcgccgtcct gcagggagga gtcctgggtc acggtcacca
     7621 cgccgccgtc ctcgaagttc atcacgcgct cccacttgaa gccctcgggg aaggacagct
     7681 tcaagtagtc ggggatgtcg gcggggtgct tcacgtaggc cttggagccg tacatgaact
     7741 gaggggacag gatgtcccag gcgaagggca gggggccgcc cttggtcacc ttcagcttgg
     7801 cggtctgggt gccctcgtag gggcggccct cgccctcgcc ctcgatctcg aactcgtggc
     7861 cgttcacgga gccctccatg tgcaccttga agcgcatgaa ctccttgatg atggccatgt
     7921 tgtcatcctc gcccttgctc accatggtgg
//
