LOCUS       sequence-178749-        7602 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  contains dll4 enhancer 2 sequence and a basal promoter driving 
            expression of eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7602)
  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 7602)
  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-basEgfp
FEATURES             Location/Qualifiers
     source          1..7602
                     /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      1393..1402
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1399..1401,1402..1404,1405..2115)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1399..1401,1402..1404,1405..2115)
                     /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(1444..1465)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1705..1724)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2052..2073
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2116..2181
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2305..2426
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(2433..2457)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2483..2503)
                     /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(2511..2527)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2543..2677
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(3116..3303,3304..3315)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3349..3365)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3349..3365)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3402..3422)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3402..3420)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3441..3457)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3454..3476)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3465..3481
                     /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(3489..3495,3496..3513,3514..3519))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3534..3555
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3672..3689)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3843..4431)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3923..3942)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4602..5393,5394..5462))
                     /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     5225..5244
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5463..5567)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5730..5751)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(6100..6111,6112..6274)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     polyA_signal    6643..6764
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6887..7597)
                     /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(6967..6990)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(7165..7184)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     7434..7452
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      join(7594..7602,1)
                     /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 caggctagac
     1321 tctagagggt atataatgga tctcgcgtct cagcctcact ttgagctcct ccacacgcta
     1381 gcgctaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     1441 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     1501 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     1561 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     1621 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     1681 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     1741 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1801 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     1861 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     1921 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1981 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     2041 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     2101 gacgagctgt acaagtccgg actcagatct cgagctcaag cttcgaattc tgcagtcgac
     2161 ggtaccgcgg gcccgggatc caccggatct agataactga tcataatcag ccataccaca
     2221 tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat
     2281 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
     2341 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     2401 ttgtccaaac tcatcaatgt atcttaacgc gtacccagct ttcttgtaca aagtggggcg
     2461 cgcctacgta actagtctcg agcaacttta ttatacatag ttgataattc actggccgtc
     2521 gttttacggt accatcgatg atgatccaga catgataaga tacattgatg agtttggaca
     2581 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     2641 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     2701 tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa
     2761 atgtggtatg gctgattatg atcctctaga tcagatctgc gaagatacgg ccacgggtgc
     2821 tcttgatcct gtggctgatt ttggactgtg ctgctcgcag ctgctgatga atcacatact
     2881 tcctccattt tcttccactg attgactgtt ataatttccc taatttccag gtcaaggtgc
     2941 tgtgcattgt ggtaatagat gtgacatgac gtcacttcca aaggaccaat gaacatgtct
     3001 gaccaatttc atataatgtg aaaacgattt tcataggcag aataaataac atttaaatta
     3061 aactgggcat cagcgcaatt caattggttt ggtaatagca agggaaaata gaatgaagtg
     3121 atctccaaaa aataagtact ttttgactgt aaataaaatt gtaaggagta aaaagtactt
     3181 ttttttctaa aaaaatgtaa ttaagtaaaa gtaaaagtat tgatttttaa ttgtactcaa
     3241 gtaaagtaaa aatccccaaa aataatactt aagtacagta atcaagtaaa attactcaag
     3301 tactttacac ctctggttct tgacccccta ccttcagcaa gcccagcaga tccactagtt
     3361 ctagagcggc cgccaccgcg gtggagctcc agcttttgtt ccctttagtg agggttaatt
     3421 gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     3481 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     3541 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     3601 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3661 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3721 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3781 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3841 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3901 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3961 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4021 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4081 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4141 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4201 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4261 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4321 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4381 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4441 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta aggcattttg
     4501 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4561 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4621 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4681 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4741 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4801 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4861 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     4921 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     4981 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5041 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     5101 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     5161 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     5221 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5281 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     5341 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     5401 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5461 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5521 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5581 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     5641 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     5701 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     5761 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccacgctggc
     5821 taagaactca tcagcctccc cggtccatct acccacgtac caatgcacca attggccaca
     5881 atgacggcta ctacatggtg ccattccttc ctctttatag gaatggagac tacctcctgt
     5941 ccaacaatgc tcttggatac gagtacgcct acctgttgga cccaggtcat tgcacaacac
     6001 cagaaatgcc ctctgatctg caaaagacgt gaatatctgt tcagacaccc atatccactc
     6061 tgttccacac aggtcagagg tttgtccagg agttcttgac agaggtgtaa aaagtactca
     6121 aaaattttac tcaagtgaaa gtacaagtac ttagggaaaa ttttactcaa ttaaaagtaa
     6181 aagtatctgg ctagaatctt acttgagtaa aagtaaaaaa gtactccatt aaaattgtac
     6241 ttgagtatta aggaagtaaa agtaaaagca agaaagaaaa ctagagattc ttgtttaagc
     6301 ttttaatctc aaaaaacatt aaatgaaatg catacaaggt tttatcctgc tttagaactg
     6361 tttgtattta attatcaaac tataagacag acaatctaat gccagtacac gctactcaaa
     6421 gttgtaaaac ctcagattta acttcagtag aagctgattc tcaaaattgt tagtgtcaag
     6481 cctagctctt ttggggctga aaagcaatcc tgcagtgctg aaaagcctct cacaggcagc
     6541 cgatgcggga agaggtgtat tagtcttgat agagaggctg caaatagcag gaaacgtgag
     6601 cagagactcc ctggtgtctg aaacacaggc ccatttacgc cttaagatac attgatgagt
     6661 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     6721 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     6781 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc
     6841 tctacaaatg tggtatggct gattatgatc tagagtcgcg gccgctttac ttgtacagct
     6901 cgtccatgcc gccggtggag tggtggccct cggcgcgctc gtactgttcc acgatggtgt
     6961 agtcctcgtt gtgggaggtg atgtccagct tgatgttgac gttgtaggcg ccgggcagct
     7021 gcacgggctt cttggccttg taggtggtct tgacctcggc gtcgtagtgg ccgccgtcct
     7081 tcagcttcag cctctgcttg atctcgccct tcagggcgcc gtcctcgggg tacatccgct
     7141 cggaggaggc ctcccagccc atggtcttct tctgcattac ggggccgtcg gaggggaagt
     7201 tggtgccgcg cagcttcacc ttgtagatga actcgccgtc ctgcagggag gagtcctggg
     7261 tcacggtcac cacgccgccg tcctcgaagt tcatcacgcg ctcccacttg aagccctcgg
     7321 ggaaggacag cttcaagtag tcggggatgt cggcggggtg cttcacgtag gccttggagc
     7381 cgtacatgaa ctgaggggac aggatgtccc aggcgaaggg cagggggccg cccttggtca
     7441 ccttcagctt ggcggtctgg gtgccctcgt aggggcggcc ctcgccctcg ccctcgatct
     7501 cgaactcgtg gccgttcacg gagccctcca tgtgcacctt gaagcgcatg aactccttga
     7561 tgatggccat gttgtcatcc tcgcccttgc tcaccatggt gg
//
