LOCUS       sequence-178729-        7517 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  contains clec14a enhancer 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 7517)
  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 7517)
  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;clec14aCE1-basEgfp
FEATURES             Location/Qualifiers
     source          1..7517
                     /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    1207..1231
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1308..1317
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1314..1316,1317..1319,1320..2030)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1314..1316,1317..1319,1320..2030)
                     /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(1359..1380)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1620..1639)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1967..1988
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2031..2096
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2220..2341
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(2348..2372)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2398..2418)
                     /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(2426..2442)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2458..2592
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(3031..3218,3219..3230)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3264..3280)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3264..3280)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3317..3337)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3317..3335)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3356..3372)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3369..3391)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3380..3396
                     /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(3404..3410,3411..3428,3429..3434))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3449..3470
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3587..3604)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3758..4346)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3838..3857)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4517..5308,5309..5377))
                     /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     5140..5159
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5378..5482)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5645..5666)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(6015..6026,6027..6189)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     polyA_signal    6558..6679
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6802..7512)
                     /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(6882..6905)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(7080..7099)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     7349..7367
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      join(7509..7517,1)
                     /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 cattgttgtt taaaaaagca
      721 gcgagctctc gtaataaaac acctgtatca ccttcatttc aactttcctt ctttgttctt
      781 tctaggcata cttgtagctc cttttcctgt tacccataat tccatttcct gtccttcttc
      841 cccacccccc tggctctcta gactgcagaa cagagagtcg agtgctcctg caacctcact
      901 tcctgccatt cagagccgtt tcctccagga agcatctgct gctctctcat gaagcggcct
      961 gttggaggaa cagaataata atacggtgtt agtcatggga cattcaacac cacattagtc
     1021 ccatgtgagc tcttgaaaac acacgcggag gtcaatcaac acgcatttga aactgacagc
     1081 gttgagtgcg acatgtaaac acagaggtat agtactactg taaagaacaa tccctgatgt
     1141 aatgagcaac gcttcctggt atttttccca gagttgtttt gttacagaca caatgaacaa
     1201 gtcaaagcaa gtttgtacaa aaaagcaggc tagactctag agggtatata atggatctcg
     1261 cgtctcagcc tcactttgag ctcctccaca cgctagcgct accggtcgcc accatggtga
     1321 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     1381 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     1441 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     1501 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     1561 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     1621 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     1681 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     1741 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     1801 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     1861 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     1921 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     1981 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag tccggactca
     2041 gatctcgagc tcaagcttcg aattctgcag tcgacggtac cgcgggcccg ggatccaccg
     2101 gatctagata actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa
     2161 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
     2221 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     2281 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     2341 aacgcgtacc cagctttctt gtacaaagtg gggcgcgcct acgtaactag tctcgagcaa
     2401 ctttattata catagttgat aattcactgg ccgtcgtttt acggtaccat cgatgatgat
     2461 ccagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     2521 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     2581 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg
     2641 tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtatggctga ttatgatcct
     2701 ctagatcaga tctgcgaaga tacggccacg ggtgctcttg atcctgtggc tgattttgga
     2761 ctgtgctgct cgcagctgct gatgaatcac atacttcctc cattttcttc cactgattga
     2821 ctgttataat ttccctaatt tccaggtcaa ggtgctgtgc attgtggtaa tagatgtgac
     2881 atgacgtcac ttccaaagga ccaatgaaca tgtctgacca atttcatata atgtgaaaac
     2941 gattttcata ggcagaataa ataacattta aattaaactg ggcatcagcg caattcaatt
     3001 ggtttggtaa tagcaaggga aaatagaatg aagtgatctc caaaaaataa gtactttttg
     3061 actgtaaata aaattgtaag gagtaaaaag tacttttttt tctaaaaaaa tgtaattaag
     3121 taaaagtaaa agtattgatt tttaattgta ctcaagtaaa gtaaaaatcc ccaaaaataa
     3181 tacttaagta cagtaatcaa gtaaaattac tcaagtactt tacacctctg gttcttgacc
     3241 ccctaccttc agcaagccca gcagatccac tagttctaga gcggccgcca ccgcggtgga
     3301 gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat
     3361 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     3421 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     3481 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     3541 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     3601 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     3661 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     3721 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     3781 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     3841 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     3901 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3961 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4021 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4081 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4141 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     4201 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4261 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4321 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4381 ctcagtggaa cgaaaactca cgttaaggca ttttggtcat gagattatca aaaaggatct
     4441 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     4501 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     4561 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     4621 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     4681 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4741 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     4801 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     4861 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     4921 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     4981 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5041 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     5101 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     5161 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     5221 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     5281 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     5341 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     5401 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     5461 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt
     5521 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     5581 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     5641 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     5701 aaaaaccgtc tatcagggcg atggcccacg ctggctaaga actcatcagc ctccccggtc
     5761 catctaccca cgtaccaatg caccaattgg ccacaatgac ggctactaca tggtgccatt
     5821 ccttcctctt tataggaatg gagactacct cctgtccaac aatgctcttg gatacgagta
     5881 cgcctacctg ttggacccag gtcattgcac aacaccagaa atgccctctg atctgcaaaa
     5941 gacgtgaata tctgttcaga cacccatatc cactctgttc cacacaggtc agaggtttgt
     6001 ccaggagttc ttgacagagg tgtaaaaagt actcaaaaat tttactcaag tgaaagtaca
     6061 agtacttagg gaaaatttta ctcaattaaa agtaaaagta tctggctaga atcttacttg
     6121 agtaaaagta aaaaagtact ccattaaaat tgtacttgag tattaaggaa gtaaaagtaa
     6181 aagcaagaaa gaaaactaga gattcttgtt taagctttta atctcaaaaa acattaaatg
     6241 aaatgcatac aaggttttat cctgctttag aactgtttgt atttaattat caaactataa
     6301 gacagacaat ctaatgccag tacacgctac tcaaagttgt aaaacctcag atttaacttc
     6361 agtagaagct gattctcaaa attgttagtg tcaagcctag ctcttttggg gctgaaaagc
     6421 aatcctgcag tgctgaaaag cctctcacag gcagccgatg cgggaagagg tgtattagtc
     6481 ttgatagaga ggctgcaaat agcaggaaac gtgagcagag actccctggt gtctgaaaca
     6541 caggcccatt tacgccttaa gatacattga tgagtttgga caaaccacaa ctagaatgca
     6601 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     6661 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     6721 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta tggctgatta
     6781 tgatctagag tcgcggccgc tttacttgta cagctcgtcc atgccgccgg tggagtggtg
     6841 gccctcggcg cgctcgtact gttccacgat ggtgtagtcc tcgttgtggg aggtgatgtc
     6901 cagcttgatg ttgacgttgt aggcgccggg cagctgcacg ggcttcttgg ccttgtaggt
     6961 ggtcttgacc tcggcgtcgt agtggccgcc gtccttcagc ttcagcctct gcttgatctc
     7021 gcccttcagg gcgccgtcct cggggtacat ccgctcggag gaggcctccc agcccatggt
     7081 cttcttctgc attacggggc cgtcggaggg gaagttggtg ccgcgcagct tcaccttgta
     7141 gatgaactcg ccgtcctgca gggaggagtc ctgggtcacg gtcaccacgc cgccgtcctc
     7201 gaagttcatc acgcgctccc acttgaagcc ctcggggaag gacagcttca agtagtcggg
     7261 gatgtcggcg gggtgcttca cgtaggcctt ggagccgtac atgaactgag gggacaggat
     7321 gtcccaggcg aagggcaggg ggccgccctt ggtcaccttc agcttggcgg tctgggtgcc
     7381 ctcgtagggg cggccctcgc cctcgccctc gatctcgaac tcgtggccgt tcacggagcc
     7441 ctccatgtgc accttgaagc gcatgaactc cttgatgatg gccatgttgt catcctcgcc
     7501 cttgctcacc atggtgg
//
