LOCUS       sequence-178699-        7604 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  contains nrp1b 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 7604)
  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 7604)
  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;nrp1bCE1-basEgfp
FEATURES             Location/Qualifiers
     source          1..7604
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     624..640
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    660..680
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    1275..1299
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1376..1385
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1382..1384,1385..1387,1388..2098)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1382..1384,1385..1387,1388..2098)
                     /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(1427..1448)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1688..1707)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2035..2056
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2099..2164
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2288..2409
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(2416..2440)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2466..2486)
                     /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(2494..2510)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2526..2660
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(3099..3286,3287..3298)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3332..3348)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3332..3348)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3385..3405)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3385..3403)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3424..3440)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3437..3459)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3448..3464
                     /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(3472..3478,3479..3496,3497..3502))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3517..3538
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3655..3672)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3826..4414)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3906..3925)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4585..5376,5377..5445))
                     /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     5208..5227
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5446..5550)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5713..5734)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(6083..6094,6095..6257)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     polyA_signal    6626..6747
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6870..7580)
                     /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(6950..6973)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(7148..7167)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     7417..7435
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      7577..7586
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 tggtaactcc ttactgtaac ttattgcacc tgatgaacaa aagatgtgaa ctgaacgcaa
       61 gtaggaggct aatagaggct agtacagtac aaagatcccc aaatgatgtg tgccggcctt
      121 agtggaggca gacccctggt tatatacctg tgctgcagaa agaggggctt acaaatggat
      181 ctctctggaa tggcattatc tcatggtggg aacagtgttg tcagcagaat cagcggggtg
      241 gactctgacc tgagaatgac agtctagcac tgaatggctc agacaatact cttgtggcca
      301 actatcaccg gaaaataagt gcctgccaaa acaaaccaga ccagatgaag ctacatggct
      361 gtagaacgtc agtatttaat gtgttgtggc cacttggttc accagattta agagctaatt
      421 tttttaaata tcacagataa tcaatcgctt ttttttttct tgactgaaaa gcttagactg
      481 gaaaattctt tgaaaaagtt tgcactacag aaaataaata cataacatcc tgcactgaat
      541 gcacactatt aatagaaaag agagcattat tgtaggaaat gtattgtata tgtacattct
      601 gtgcatttca caattttctc gagcaggaaa cagctatgac catgattacg ccaagctatc
      661 aactttgtat agaaaagttg gggatgtgct gaaaatgtct ctaacgcacc tatgtgacgt
      721 gctttaacta ggttaacgat ggtgaacggt cggcacggct cattaatagg ccttcacgtc
      781 cacttaaaga tggctccatc agaaagagag gtagaagagt ccttgtaacg tccttctccc
      841 tcgttcctcc atttgtcttc tagttttatc ttgctcaagc tgaaggggaa gccctgcgct
      901 gcgggaccgc cgctgtttta ccttcgaccc accgctatat ttatcccatt gtgctgaact
      961 aattgctggt ctgacatcac ctcttgaaga aggacagctg atgaacaaag catctgcgga
     1021 ttatttttag tttgttctta agacggagct tcagggacac acaggcagag agagagagag
     1081 acaggaagtg ctcgtgcagc tggagacaat gttatcttgg tagcaggaca ggaaatgggg
     1141 tccaatcact tcctggggcc tgagagcttt gctaagacac tggtttgagt tctgatcggc
     1201 tgtttttcca cctcaagggt ttcagcacaa gggggtttga gaagtagctt tttagtgctg
     1261 ataatcagat ttagtcaagt ttgtacaaaa aagcaggcta gactctagag ggtatataat
     1321 ggatctcgcg tctcagcctc actttgagct cctccacacg ctagcgctac cggtcgccac
     1381 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     1441 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     1501 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     1561 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     1621 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     1681 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1741 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1801 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     1861 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     1921 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1981 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     2041 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagtc
     2101 cggactcaga tctcgagctc aagcttcgaa ttctgcagtc gacggtaccg cgggcccggg
     2161 atccaccgga tctagataac tgatcataat cagccatacc acatttgtag aggttttact
     2221 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     2281 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     2341 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     2401 tgtatcttaa cgcgtaccca gctttcttgt acaaagtggg gcgcgcctac gtaactagtc
     2461 tcgagcaact ttattataca tagttgataa ttcactggcc gtcgttttac ggtaccatcg
     2521 atgatgatcc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     2581 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     2641 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     2701 gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt atggctgatt
     2761 atgatcctct agatcagatc tgcgaagata cggccacggg tgctcttgat cctgtggctg
     2821 attttggact gtgctgctcg cagctgctga tgaatcacat acttcctcca ttttcttcca
     2881 ctgattgact gttataattt ccctaatttc caggtcaagg tgctgtgcat tgtggtaata
     2941 gatgtgacat gacgtcactt ccaaaggacc aatgaacatg tctgaccaat ttcatataat
     3001 gtgaaaacga ttttcatagg cagaataaat aacatttaaa ttaaactggg catcagcgca
     3061 attcaattgg tttggtaata gcaagggaaa atagaatgaa gtgatctcca aaaaataagt
     3121 actttttgac tgtaaataaa attgtaagga gtaaaaagta cttttttttc taaaaaaatg
     3181 taattaagta aaagtaaaag tattgatttt taattgtact caagtaaagt aaaaatcccc
     3241 aaaaataata cttaagtaca gtaatcaagt aaaattactc aagtacttta cacctctggt
     3301 tcttgacccc ctaccttcag caagcccagc agatccacta gttctagagc ggccgccacc
     3361 gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc
     3421 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     3481 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     3541 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     3601 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     3661 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     3721 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     3781 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3841 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3901 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3961 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4021 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4081 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4141 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4201 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4261 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4321 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4381 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     4441 gtctgacgct cagtggaacg aaaactcacg ttaaggcatt ttggtcatga gattatcaaa
     4501 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     4561 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     4621 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     4681 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     4741 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4801 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4861 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     4921 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     4981 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5041 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     5101 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5161 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     5221 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5281 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5341 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     5401 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     5461 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     5521 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt
     5581 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     5641 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     5701 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     5761 aaagggcgaa aaaccgtcta tcagggcgat ggcccacgct ggctaagaac tcatcagcct
     5821 ccccggtcca tctacccacg taccaatgca ccaattggcc acaatgacgg ctactacatg
     5881 gtgccattcc ttcctcttta taggaatgga gactacctcc tgtccaacaa tgctcttgga
     5941 tacgagtacg cctacctgtt ggacccaggt cattgcacaa caccagaaat gccctctgat
     6001 ctgcaaaaga cgtgaatatc tgttcagaca cccatatcca ctctgttcca cacaggtcag
     6061 aggtttgtcc aggagttctt gacagaggtg taaaaagtac tcaaaaattt tactcaagtg
     6121 aaagtacaag tacttaggga aaattttact caattaaaag taaaagtatc tggctagaat
     6181 cttacttgag taaaagtaaa aaagtactcc attaaaattg tacttgagta ttaaggaagt
     6241 aaaagtaaaa gcaagaaaga aaactagaga ttcttgttta agcttttaat ctcaaaaaac
     6301 attaaatgaa atgcatacaa ggttttatcc tgctttagaa ctgtttgtat ttaattatca
     6361 aactataaga cagacaatct aatgccagta cacgctactc aaagttgtaa aacctcagat
     6421 ttaacttcag tagaagctga ttctcaaaat tgttagtgtc aagcctagct cttttggggc
     6481 tgaaaagcaa tcctgcagtg ctgaaaagcc tctcacaggc agccgatgcg ggaagaggtg
     6541 tattagtctt gatagagagg ctgcaaatag caggaaacgt gagcagagac tccctggtgt
     6601 ctgaaacaca ggcccattta cgccttaaga tacattgatg agtttggaca aaccacaact
     6661 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     6721 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     6781 gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg
     6841 gctgattatg atctagagtc gcggccgctt tacttgtaca gctcgtccat gccgccggtg
     6901 gagtggtggc cctcggcgcg ctcgtactgt tccacgatgg tgtagtcctc gttgtgggag
     6961 gtgatgtcca gcttgatgtt gacgttgtag gcgccgggca gctgcacggg cttcttggcc
     7021 ttgtaggtgg tcttgacctc ggcgtcgtag tggccgccgt ccttcagctt cagcctctgc
     7081 ttgatctcgc ccttcagggc gccgtcctcg gggtacatcc gctcggagga ggcctcccag
     7141 cccatggtct tcttctgcat tacggggccg tcggagggga agttggtgcc gcgcagcttc
     7201 accttgtaga tgaactcgcc gtcctgcagg gaggagtcct gggtcacggt caccacgccg
     7261 ccgtcctcga agttcatcac gcgctcccac ttgaagccct cggggaagga cagcttcaag
     7321 tagtcgggga tgtcggcggg gtgcttcacg taggccttgg agccgtacat gaactgaggg
     7381 gacaggatgt cccaggcgaa gggcaggggg ccgcccttgg tcaccttcag cttggcggtc
     7441 tgggtgccct cgtaggggcg gccctcgccc tcgccctcga tctcgaactc gtggccgttc
     7501 acggagccct ccatgtgcac cttgaagcgc atgaactcct tgatgatggc catgttgtca
     7561 tcctcgccct tgctcaccat ggtggcgacc ggtaatgtca gacc
//
