LOCUS       sequence-178934-        7345 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  contains cldn5b specific gene promoter driving expression of eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7345)
  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 7345)
  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;cldn5bP1Egfp
FEATURES             Location/Qualifiers
     source          1..7345
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     628..644
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    664..684
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     686..705
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        686..704
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    713..820
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     729..745
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     730..746
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(780..796)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(833..851)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    851..875
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1352..1361
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1358..1360,1361..1363,1364..2074)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1358..1360,1361..1363,1364..2074)
                     /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(1403..1424)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1664..1683)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2011..2032
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2075..2140
                     /label=MCS
                     /note="multiple cloning site"
     protein_bind    complement(2161..2185)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2211..2231)
                     /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(2239..2255)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2271..2405
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(2844..3031,3032..3043)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3077..3093)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(3130..3148)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3169..3185)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3182..3204)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3193..3209
                     /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(3217..3223,3224..3241,3242..3247))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3262..3283
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3400..3417)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3571..4159)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3651..3670)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4330..5121,5122..5190))
                     /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     4953..4972
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5191..5295)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5458..5479)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(5828..5839,5840..6002)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     polyA_signal    6371..6492
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6615..7325)
                     /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(6695..6718)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(6893..6912)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     7162..7180
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      7322..7331
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 gacctggtaa ctccttactg taacttattg cacctgatga acaaaagatg tgaactgaac
       61 gcaagtagga ggctaataga ggctagtaca gtacaaagat ccccaaatga tgtgtgccgg
      121 ccttagtgga ggcagacccc tggttatata cctgtgctgc agaaagaggg gcttacaaat
      181 ggatctctct ggaatggcat tatctcatgg tgggaacagt gttgtcagca gaatcagcgg
      241 ggtggactct gacctgagaa tgacagtcta gcactgaatg gctcagacaa tactcttgtg
      301 gccaactatc accggaaaat aagtgcctgc caaaacaaac cagaccagat gaagctacat
      361 ggctgtagaa cgtcagtatt taatgtgttg tggccacttg gttcaccaga tttaagagct
      421 aattttttta aatatcacag ataatcaatc gctttttttt ttcttgactg aaaagcttag
      481 actggaaaat tctttgaaaa agtttgcact acagaaaata aatacataac atcctgcact
      541 gaatgcacac tattaataga aaagagagca ttattgtagg aaatgtattg tatatgtaca
      601 ttctgtgcat ttcacaattt tctcgagcag gaaacagcta tgaccatgat tacgccaagc
      661 tatcaacttt gtatagaaaa gttggtaata cgactcacta tagggcgaat tgggtaccgg
      721 gccccccctc gaggtcgacg gtatcgataa gcttgatatc gaattcctgc agcccggggg
      781 atccactagt tctagagcgg ccgccaccgc ggtggagctc cagcttttgt tccctttagt
      841 gagggttaat tcaagtttgt acaaaaaagc aggctcgcta aattaaacta agcgttattc
      901 gagacgtggc cgctttaacg ttacctcagg tgcgcgttca ttatcagcgc tccgtaacaa
      961 actattcctg ccctggctgc cttacaagat cacctaattc tctgttgttt tacacatccg
     1021 tgtgcctgca ttgacacttg cccctcttct gtaaaggggg cttttcttcc taaagaatgc
     1081 caggaaatca cacttccagt agcaactacc gtcacgttta aaaaactctg actgcgggat
     1141 gctaattttt ttgttcggct gttctaactt gtgattggct gctggcagac gcgccgcgct
     1201 ctgattggct ggagccgctc ctctttgttg cgggcgctct ataacagcat cctcccttca
     1261 ttgcagagca gaaactcctc aagcacttca tttgagcgat gtagagacac ttctgctgct
     1321 tctgacagat ttagagtttg gagaaaaaat cgccaccatg gtgagcaagg gcgaggagct
     1381 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     1441 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     1501 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     1561 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     1621 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     1681 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     1741 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     1801 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     1861 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     1921 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     1981 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     2041 cgggatcact ctcggcatgg acgagctgta caagtccgga ctcagatctc gagctcaagc
     2101 ttcgaattct gcagtcgacg gtaccgcggg cccgggatcc accggatcta gataactgat
     2161 acccagcttt cttgtacaaa gtggggcgcg cctacgtaac tagtctcgag caactttatt
     2221 atacatagtt gataattcac tggccgtcgt tttacggtac catcgatgat gatccagaca
     2281 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     2341 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     2401 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg
     2461 ttttttaaag caagtaaaac ctctacaaat gtggtatggc tgattatgat cctctagatc
     2521 agatctgcga agatacggcc acgggtgctc ttgatcctgt ggctgatttt ggactgtgct
     2581 gctcgcagct gctgatgaat cacatacttc ctccattttc ttccactgat tgactgttat
     2641 aatttcccta atttccaggt caaggtgctg tgcattgtgg taatagatgt gacatgacgt
     2701 cacttccaaa ggaccaatga acatgtctga ccaatttcat ataatgtgaa aacgattttc
     2761 ataggcagaa taaataacat ttaaattaaa ctgggcatca gcgcaattca attggtttgg
     2821 taatagcaag ggaaaataga atgaagtgat ctccaaaaaa taagtacttt ttgactgtaa
     2881 ataaaattgt aaggagtaaa aagtactttt ttttctaaaa aaatgtaatt aagtaaaagt
     2941 aaaagtattg atttttaatt gtactcaagt aaagtaaaaa tccccaaaaa taatacttaa
     3001 gtacagtaat caagtaaaat tactcaagta ctttacacct ctggttcttg accccctacc
     3061 ttcagcaagc ccagcagatc cactagttct agagcggccg ccaccgcggt ggagctccag
     3121 cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt
     3181 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     3241 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     3301 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     3361 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     3421 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     3481 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     3541 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     3601 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     3661 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3721 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3781 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3841 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3901 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3961 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     4021 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4081 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4141 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4201 gaacgaaaac tcacgttaag gcattttggt catgagatta tcaaaaagga tcttcaccta
     4261 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     4321 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     4381 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     4441 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     4501 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     4561 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     4621 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     4681 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     4741 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     4801 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     4861 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     4921 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     4981 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     5041 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     5101 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     5161 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     5221 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5281 aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt
     5341 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     5401 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     5461 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     5521 gtctatcagg gcgatggccc acgctggcta agaactcatc agcctccccg gtccatctac
     5581 ccacgtacca atgcaccaat tggccacaat gacggctact acatggtgcc attccttcct
     5641 ctttatagga atggagacta cctcctgtcc aacaatgctc ttggatacga gtacgcctac
     5701 ctgttggacc caggtcattg cacaacacca gaaatgccct ctgatctgca aaagacgtga
     5761 atatctgttc agacacccat atccactctg ttccacacag gtcagaggtt tgtccaggag
     5821 ttcttgacag aggtgtaaaa agtactcaaa aattttactc aagtgaaagt acaagtactt
     5881 agggaaaatt ttactcaatt aaaagtaaaa gtatctggct agaatcttac ttgagtaaaa
     5941 gtaaaaaagt actccattaa aattgtactt gagtattaag gaagtaaaag taaaagcaag
     6001 aaagaaaact agagattctt gtttaagctt ttaatctcaa aaaacattaa atgaaatgca
     6061 tacaaggttt tatcctgctt tagaactgtt tgtatttaat tatcaaacta taagacagac
     6121 aatctaatgc cagtacacgc tactcaaagt tgtaaaacct cagatttaac ttcagtagaa
     6181 gctgattctc aaaattgtta gtgtcaagcc tagctctttt ggggctgaaa agcaatcctg
     6241 cagtgctgaa aagcctctca caggcagccg atgcgggaag aggtgtatta gtcttgatag
     6301 agaggctgca aatagcagga aacgtgagca gagactccct ggtgtctgaa acacaggccc
     6361 atttacgcct taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     6421 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     6481 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg
     6541 tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtatggctga ttatgatcta
     6601 gagtcgcggc cgctttactt gtacagctcg tccatgccgc cggtggagtg gtggccctcg
     6661 gcgcgctcgt actgttccac gatggtgtag tcctcgttgt gggaggtgat gtccagcttg
     6721 atgttgacgt tgtaggcgcc gggcagctgc acgggcttct tggccttgta ggtggtcttg
     6781 acctcggcgt cgtagtggcc gccgtccttc agcttcagcc tctgcttgat ctcgcccttc
     6841 agggcgccgt cctcggggta catccgctcg gaggaggcct cccagcccat ggtcttcttc
     6901 tgcattacgg ggccgtcgga ggggaagttg gtgccgcgca gcttcacctt gtagatgaac
     6961 tcgccgtcct gcagggagga gtcctgggtc acggtcacca cgccgccgtc ctcgaagttc
     7021 atcacgcgct cccacttgaa gccctcgggg aaggacagct tcaagtagtc ggggatgtcg
     7081 gcggggtgct tcacgtaggc cttggagccg tacatgaact gaggggacag gatgtcccag
     7141 gcgaagggca gggggccgcc cttggtcacc ttcagcttgg cggtctgggt gccctcgtag
     7201 gggcggccct cgccctcgcc ctcgatctcg aactcgtggc cgttcacgga gccctccatg
     7261 tgcaccttga agcgcatgaa ctccttgatg atggccatgt tgtcatcctc gcccttgctc
     7321 accatggtgg cgaccggtaa tgtca
//
