LOCUS       sequence-178703-        7324 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  contains cldn5b 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 7324)
  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 7324)
  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-basEgfp
FEATURES             Location/Qualifiers
     source          1..7324
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     631..647
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    667..687
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    1002..1026
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1103..1112
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1109..1111,1112..1114,1115..1825)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1109..1111,1112..1114,1115..1825)
                     /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(1154..1175)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1415..1434)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1762..1783
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1826..1891
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2015..2136
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(2143..2167)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2193..2213)
                     /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(2221..2237)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2253..2387
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(2826..3013,3014..3025)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3059..3075)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3059..3075)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3112..3132)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3112..3130)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3151..3167)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3164..3186)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3175..3191
                     /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(3199..3205,3206..3223,3224..3229))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3244..3265
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3382..3399)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3553..4141)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3633..3652)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4312..5103,5104..5172))
                     /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     4935..4954
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5173..5277)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5440..5461)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(5810..5821,5822..5984)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     polyA_signal    6353..6474
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6597..7307)
                     /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(6677..6700)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(6875..6894)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     7144..7162
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      7304..7313
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 tcagacctgg taactcctta ctgtaactta ttgcacctga tgaacaaaag atgtgaactg
       61 aacgcaagta ggaggctaat agaggctagt acagtacaaa gatccccaaa tgatgtgtgc
      121 cggccttagt ggaggcagac ccctggttat atacctgtgc tgcagaaaga ggggcttaca
      181 aatggatctc tctggaatgg cattatctca tggtgggaac agtgttgtca gcagaatcag
      241 cggggtggac tctgacctga gaatgacagt ctagcactga atggctcaga caatactctt
      301 gtggccaact atcaccggaa aataagtgcc tgccaaaaca aaccagacca gatgaagcta
      361 catggctgta gaacgtcagt atttaatgtg ttgtggccac ttggttcacc agatttaaga
      421 gctaattttt ttaaatatca cagataatca atcgcttttt tttttcttga ctgaaaagct
      481 tagactggaa aattctttga aaaagtttgc actacagaaa ataaatacat aacatcctgc
      541 actgaatgca cactattaat agaaaagaga gcattattgt aggaaatgta ttgtatatgt
      601 acattctgtg catttcacaa ttttctcgag caggaaacag ctatgaccat gattacgcca
      661 agctatcaac tttgtataga aaagttgcag gatttcacac tatacaactc catctgtcca
      721 cagcccacgt aactccgacc ggctcaaatc tacatccaca ggtcgagatc ctccacgccg
      781 tgccagagga ggaggtctgg atgctttgtt ttctgtgttc acgtcctgcc caatgtcagc
      841 gatgaggtcg gatagatacg tgttgaatcc cagacacctg gcgtgcagtg agctcacacc
      901 agaggaaaca tctgcagaga acatgcagat cagagcttct gtgcacttct gccgtctgtc
      961 tggagagcac tcaggattcc tgcttatgtt atgtactgta tgcaagtttg tacaaaaaag
     1021 caggctagac tctagagggt atataatgga tctcgcgtct cagcctcact ttgagctcct
     1081 ccacacgcta gcgctaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg
     1141 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     1201 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     1261 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     1321 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     1381 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     1441 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     1501 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     1561 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     1621 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     1681 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     1741 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     1801 tctcggcatg gacgagctgt acaagtccgg actcagatct cgagctcaag cttcgaattc
     1861 tgcagtcgac ggtaccgcgg gcccgggatc caccggatct agataactga tcataatcag
     1921 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa
     1981 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg
     2041 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     2101 tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc gtacccagct ttcttgtaca
     2161 aagtggggcg cgcctacgta actagtctcg agcaacttta ttatacatag ttgataattc
     2221 actggccgtc gttttacggt accatcgatg atgatccaga catgataaga tacattgatg
     2281 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     2341 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     2401 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa
     2461 acctctacaa atgtggtatg gctgattatg atcctctaga tcagatctgc gaagatacgg
     2521 ccacgggtgc tcttgatcct gtggctgatt ttggactgtg ctgctcgcag ctgctgatga
     2581 atcacatact tcctccattt tcttccactg attgactgtt ataatttccc taatttccag
     2641 gtcaaggtgc tgtgcattgt ggtaatagat gtgacatgac gtcacttcca aaggaccaat
     2701 gaacatgtct gaccaatttc atataatgtg aaaacgattt tcataggcag aataaataac
     2761 atttaaatta aactgggcat cagcgcaatt caattggttt ggtaatagca agggaaaata
     2821 gaatgaagtg atctccaaaa aataagtact ttttgactgt aaataaaatt gtaaggagta
     2881 aaaagtactt ttttttctaa aaaaatgtaa ttaagtaaaa gtaaaagtat tgatttttaa
     2941 ttgtactcaa gtaaagtaaa aatccccaaa aataatactt aagtacagta atcaagtaaa
     3001 attactcaag tactttacac ctctggttct tgacccccta ccttcagcaa gcccagcaga
     3061 tccactagtt ctagagcggc cgccaccgcg gtggagctcc agcttttgtt ccctttagtg
     3121 agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     3181 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     3241 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     3301 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     3361 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     3421 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     3481 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     3541 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     3601 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     3661 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     3721 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     3781 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     3841 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     3901 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     3961 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4021 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4081 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4141 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4201 aggcattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     4261 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     4321 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     4381 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     4441 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     4501 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     4561 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     4621 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     4681 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     4741 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     4801 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     4861 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     4921 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     4981 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5041 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5101 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5161 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5221 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5281 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta
     5341 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     5401 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     5461 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     5521 ccacgctggc taagaactca tcagcctccc cggtccatct acccacgtac caatgcacca
     5581 attggccaca atgacggcta ctacatggtg ccattccttc ctctttatag gaatggagac
     5641 tacctcctgt ccaacaatgc tcttggatac gagtacgcct acctgttgga cccaggtcat
     5701 tgcacaacac cagaaatgcc ctctgatctg caaaagacgt gaatatctgt tcagacaccc
     5761 atatccactc tgttccacac aggtcagagg tttgtccagg agttcttgac agaggtgtaa
     5821 aaagtactca aaaattttac tcaagtgaaa gtacaagtac ttagggaaaa ttttactcaa
     5881 ttaaaagtaa aagtatctgg ctagaatctt acttgagtaa aagtaaaaaa gtactccatt
     5941 aaaattgtac ttgagtatta aggaagtaaa agtaaaagca agaaagaaaa ctagagattc
     6001 ttgtttaagc ttttaatctc aaaaaacatt aaatgaaatg catacaaggt tttatcctgc
     6061 tttagaactg tttgtattta attatcaaac tataagacag acaatctaat gccagtacac
     6121 gctactcaaa gttgtaaaac ctcagattta acttcagtag aagctgattc tcaaaattgt
     6181 tagtgtcaag cctagctctt ttggggctga aaagcaatcc tgcagtgctg aaaagcctct
     6241 cacaggcagc cgatgcggga agaggtgtat tagtcttgat agagaggctg caaatagcag
     6301 gaaacgtgag cagagactcc ctggtgtctg aaacacaggc ccatttacgc cttaagatac
     6361 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     6421 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     6481 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc
     6541 aagtaaaacc tctacaaatg tggtatggct gattatgatc tagagtcgcg gccgctttac
     6601 ttgtacagct cgtccatgcc gccggtggag tggtggccct cggcgcgctc gtactgttcc
     6661 acgatggtgt agtcctcgtt gtgggaggtg atgtccagct tgatgttgac gttgtaggcg
     6721 ccgggcagct gcacgggctt cttggccttg taggtggtct tgacctcggc gtcgtagtgg
     6781 ccgccgtcct tcagcttcag cctctgcttg atctcgccct tcagggcgcc gtcctcgggg
     6841 tacatccgct cggaggaggc ctcccagccc atggtcttct tctgcattac ggggccgtcg
     6901 gaggggaagt tggtgccgcg cagcttcacc ttgtagatga actcgccgtc ctgcagggag
     6961 gagtcctggg tcacggtcac cacgccgccg tcctcgaagt tcatcacgcg ctcccacttg
     7021 aagccctcgg ggaaggacag cttcaagtag tcggggatgt cggcggggtg cttcacgtag
     7081 gccttggagc cgtacatgaa ctgaggggac aggatgtccc aggcgaaggg cagggggccg
     7141 cccttggtca ccttcagctt ggcggtctgg gtgccctcgt aggggcggcc ctcgccctcg
     7201 ccctcgatct cgaactcgtg gccgttcacg gagccctcca tgtgcacctt gaagcgcatg
     7261 aactccttga tgatggccat gttgtcatcc tcgcccttgc tcaccatggt ggcgaccggt
     7321 aatg
//
