LOCUS       sequence-178874-        7493 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  contains cldn5b enhancer sequence, and cldn5b specific gene promoter
            driving expression of eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7493)
  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 7493)
  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;cldn5bCE1-P1Egfp
FEATURES             Location/Qualifiers
     source          1..7493
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     633..649
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    669..689
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    1004..1028
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1505..1514
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1511..1513,1514..1516,1517..2227)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1511..1513,1514..1516,1517..2227)
                     /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(1556..1577)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1817..1836)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2164..2185
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2228..2293
                     /label=MCS
                     /note="multiple cloning site"
     protein_bind    complement(2314..2338)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2364..2384)
                     /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(2392..2408)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2424..2558
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(2997..3184,3185..3196)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3230..3246)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3230..3246)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3283..3303)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3283..3301)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3322..3338)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3335..3357)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3346..3362
                     /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(3370..3376,3377..3394,3395..3400))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3415..3436
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3553..3570)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3724..4312)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3804..3823)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4483..5274,5275..5343))
                     /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     5106..5125
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5344..5448)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5611..5632)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(5981..5992,5993..6155)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     polyA_signal    6524..6645
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6768..7478)
                     /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(6848..6871)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(7046..7065)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     7315..7333
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      7475..7484
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 tgtcagacct ggtaactcct tactgtaact tattgcacct gatgaacaaa agatgtgaac
       61 tgaacgcaag taggaggcta atagaggcta gtacagtaca aagatcccca aatgatgtgt
      121 gccggcctta gtggaggcag acccctggtt atatacctgt gctgcagaaa gaggggctta
      181 caaatggatc tctctggaat ggcattatct catggtggga acagtgttgt cagcagaatc
      241 agcggggtgg actctgacct gagaatgaca gtctagcact gaatggctca gacaatactc
      301 ttgtggccaa ctatcaccgg aaaataagtg cctgccaaaa caaaccagac cagatgaagc
      361 tacatggctg tagaacgtca gtatttaatg tgttgtggcc acttggttca ccagatttaa
      421 gagctaattt ttttaaatat cacagataat caatcgcttt tttttttctt gactgaaaag
      481 cttagactgg aaaattcttt gaaaaagttt gcactacaga aaataaatac ataacatcct
      541 gcactgaatg cacactatta atagaaaaga gagcattatt gtaggaaatg tattgtatat
      601 gtacattctg tgcatttcac aattttctcg agcaggaaac agctatgacc atgattacgc
      661 caagctatca actttgtata gaaaagttgc aggatttcac actatacaac tccatctgtc
      721 cacagcccac gtaactccga ccggctcaaa tctacatcca caggtcgaga tcctccacgc
      781 cgtgccagag gaggaggtct ggatgctttg ttttctgtgt tcacgtcctg cccaatgtca
      841 gcgatgaggt cggatagata cgtgttgaat cccagacacc tggcgtgcag tgagctcaca
      901 ccagaggaaa catctgcaga gaacatgcag atcagagctt ctgtgcactt ctgccgtctg
      961 tctggagagc actcaggatt cctgcttatg ttatgtactg tatgcaagtt tgtacaaaaa
     1021 agcaggctcg ctaaattaaa ctaagcgtta ttcgagacgt ggccgcttta acgttacctc
     1081 aggtgcgcgt tcattatcag cgctccgtaa caaactattc ctgccctggc tgccttacaa
     1141 gatcacctaa ttctctgttg ttttacacat ccgtgtgcct gcattgacac ttgcccctct
     1201 tctgtaaagg gggcttttct tcctaaagaa tgccaggaaa tcacacttcc agtagcaact
     1261 accgtcacgt ttaaaaaact ctgactgcgg gatgctaatt tttttgttcg gctgttctaa
     1321 cttgtgattg gctgctggca gacgcgccgc gctctgattg gctggagccg ctcctctttg
     1381 ttgcgggcgc tctataacag catcctccct tcattgcaga gcagaaactc ctcaagcact
     1441 tcatttgagc gatgtagaga cacttctgct gcttctgaca gatttagagt ttggagaaaa
     1501 aatcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     1561 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     1621 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     1681 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     1741 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     1801 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     1861 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     1921 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     1981 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     2041 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     2101 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     2161 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     2221 gtacaagtcc ggactcagat ctcgagctca agcttcgaat tctgcagtcg acggtaccgc
     2281 gggcccggga tccaccggat ctagataact gatacccagc tttcttgtac aaagtggggc
     2341 gcgcctacgt aactagtctc gagcaacttt attatacata gttgataatt cactggccgt
     2401 cgttttacgg taccatcgat gatgatccag acatgataag atacattgat gagtttggac
     2461 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     2521 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     2581 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca
     2641 aatgtggtat ggctgattat gatcctctag atcagatctg cgaagatacg gccacgggtg
     2701 ctcttgatcc tgtggctgat tttggactgt gctgctcgca gctgctgatg aatcacatac
     2761 ttcctccatt ttcttccact gattgactgt tataatttcc ctaatttcca ggtcaaggtg
     2821 ctgtgcattg tggtaataga tgtgacatga cgtcacttcc aaaggaccaa tgaacatgtc
     2881 tgaccaattt catataatgt gaaaacgatt ttcataggca gaataaataa catttaaatt
     2941 aaactgggca tcagcgcaat tcaattggtt tggtaatagc aagggaaaat agaatgaagt
     3001 gatctccaaa aaataagtac tttttgactg taaataaaat tgtaaggagt aaaaagtact
     3061 tttttttcta aaaaaatgta attaagtaaa agtaaaagta ttgattttta attgtactca
     3121 agtaaagtaa aaatccccaa aaataatact taagtacagt aatcaagtaa aattactcaa
     3181 gtactttaca cctctggttc ttgaccccct accttcagca agcccagcag atccactagt
     3241 tctagagcgg ccgccaccgc ggtggagctc cagcttttgt tccctttagt gagggttaat
     3301 tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     3361 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     3421 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3481 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3541 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3601 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3661 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3721 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3781 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3841 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3901 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3961 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4021 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4081 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4141 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4201 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4261 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4321 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aaggcatttt
     4381 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4441 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4501 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4561 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4621 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4681 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4741 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     4801 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     4861 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     4921 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     4981 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5041 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5101 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5161 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5221 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5281 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5341 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5401 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5461 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     5521 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     5581 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     5641 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccacgctgg
     5701 ctaagaactc atcagcctcc ccggtccatc tacccacgta ccaatgcacc aattggccac
     5761 aatgacggct actacatggt gccattcctt cctctttata ggaatggaga ctacctcctg
     5821 tccaacaatg ctcttggata cgagtacgcc tacctgttgg acccaggtca ttgcacaaca
     5881 ccagaaatgc cctctgatct gcaaaagacg tgaatatctg ttcagacacc catatccact
     5941 ctgttccaca caggtcagag gtttgtccag gagttcttga cagaggtgta aaaagtactc
     6001 aaaaatttta ctcaagtgaa agtacaagta cttagggaaa attttactca attaaaagta
     6061 aaagtatctg gctagaatct tacttgagta aaagtaaaaa agtactccat taaaattgta
     6121 cttgagtatt aaggaagtaa aagtaaaagc aagaaagaaa actagagatt cttgtttaag
     6181 cttttaatct caaaaaacat taaatgaaat gcatacaagg ttttatcctg ctttagaact
     6241 gtttgtattt aattatcaaa ctataagaca gacaatctaa tgccagtaca cgctactcaa
     6301 agttgtaaaa cctcagattt aacttcagta gaagctgatt ctcaaaattg ttagtgtcaa
     6361 gcctagctct tttggggctg aaaagcaatc ctgcagtgct gaaaagcctc tcacaggcag
     6421 ccgatgcggg aagaggtgta ttagtcttga tagagaggct gcaaatagca ggaaacgtga
     6481 gcagagactc cctggtgtct gaaacacagg cccatttacg ccttaagata cattgatgag
     6541 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     6601 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     6661 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
     6721 ctctacaaat gtggtatggc tgattatgat ctagagtcgc ggccgcttta cttgtacagc
     6781 tcgtccatgc cgccggtgga gtggtggccc tcggcgcgct cgtactgttc cacgatggtg
     6841 tagtcctcgt tgtgggaggt gatgtccagc ttgatgttga cgttgtaggc gccgggcagc
     6901 tgcacgggct tcttggcctt gtaggtggtc ttgacctcgg cgtcgtagtg gccgccgtcc
     6961 ttcagcttca gcctctgctt gatctcgccc ttcagggcgc cgtcctcggg gtacatccgc
     7021 tcggaggagg cctcccagcc catggtcttc ttctgcatta cggggccgtc ggaggggaag
     7081 ttggtgccgc gcagcttcac cttgtagatg aactcgccgt cctgcaggga ggagtcctgg
     7141 gtcacggtca ccacgccgcc gtcctcgaag ttcatcacgc gctcccactt gaagccctcg
     7201 gggaaggaca gcttcaagta gtcggggatg tcggcggggt gcttcacgta ggccttggag
     7261 ccgtacatga actgagggga caggatgtcc caggcgaagg gcagggggcc gcccttggtc
     7321 accttcagct tggcggtctg ggtgccctcg taggggcggc cctcgccctc gccctcgatc
     7381 tcgaactcgt ggccgttcac ggagccctcc atgtgcacct tgaagcgcat gaactccttg
     7441 atgatggcca tgttgtcatc ctcgcccttg ctcaccatgg tggcgaccgg taa
//
