LOCUS       sequence-178974-        7176 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  contains a basal promoter driving expression of eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7176)
  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 7176)
  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;basPEgfp
FEATURES             Location/Qualifiers
     source          1..7176
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      92..101
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(98..100,101..103,104..814)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(98..100,101..103,104..814)
                     /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(143..164)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(404..423)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     751..772
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    815..880
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    1004..1125
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(1132..1156)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1182..1202)
                     /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(1210..1226)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    1242..1376
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(1815..2002,2003..2014)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(2048..2064)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(2101..2119)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2140..2156)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2153..2175)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2164..2180
                     /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(2188..2194,2195..2212,2213..2218))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2233..2254
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2371..2388)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2542..3130)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2622..2641)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3301..4092,4093..4161))
                     /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     3924..3943
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4162..4266)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4429..4450)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(4799..4810,4811..4973)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     polyA_signal    5342..5463
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5586..6296)
                     /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(5666..5689)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(5864..5883)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     6133..6151
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      6293..6302
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     6944..6960
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6980..7000
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     7002..7021
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7002..7020
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    7029..7136
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     7045..7061
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     7046..7062
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7096..7112)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(7149..7167)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    join(7167..7176,1..15)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 acaaaaaagc aggctagact ctagagggta tataatggat ctcgcgtctc agcctcactt
       61 tgagctcctc cacacgctag cgctaccggt cgccaccatg gtgagcaagg gcgaggagct
      121 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
      181 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
      241 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
      301 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
      361 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
      421 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
      481 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
      541 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
      601 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
      661 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
      721 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
      781 cgggatcact ctcggcatgg acgagctgta caagtccgga ctcagatctc gagctcaagc
      841 ttcgaattct gcagtcgacg gtaccgcggg cccgggatcc accggatcta gataactgat
      901 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
      961 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     1021 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     1081 actgcattct agttgtggtt tgtccaaact catcaatgta tcttaacgcg tacccagctt
     1141 tcttgtacaa agtggggcgc gcctacgtaa ctagtctcga gcaactttat tatacatagt
     1201 tgataattca ctggccgtcg ttttacggta ccatcgatga tgatccagac atgataagat
     1261 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     1321 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     1381 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa
     1441 gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tcctctagat cagatctgcg
     1501 aagatacggc cacgggtgct cttgatcctg tggctgattt tggactgtgc tgctcgcagc
     1561 tgctgatgaa tcacatactt cctccatttt cttccactga ttgactgtta taatttccct
     1621 aatttccagg tcaaggtgct gtgcattgtg gtaatagatg tgacatgacg tcacttccaa
     1681 aggaccaatg aacatgtctg accaatttca tataatgtga aaacgatttt cataggcaga
     1741 ataaataaca tttaaattaa actgggcatc agcgcaattc aattggtttg gtaatagcaa
     1801 gggaaaatag aatgaagtga tctccaaaaa ataagtactt tttgactgta aataaaattg
     1861 taaggagtaa aaagtacttt tttttctaaa aaaatgtaat taagtaaaag taaaagtatt
     1921 gatttttaat tgtactcaag taaagtaaaa atccccaaaa ataatactta agtacagtaa
     1981 tcaagtaaaa ttactcaagt actttacacc tctggttctt gaccccctac cttcagcaag
     2041 cccagcagat ccactagttc tagagcggcc gccaccgcgg tggagctcca gcttttgttc
     2101 cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     2161 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     2221 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     2281 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     2341 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     2401 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     2461 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     2521 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     2581 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     2641 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     2701 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     2761 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     2821 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     2881 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     2941 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     3001 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     3061 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     3121 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     3181 ctcacgttaa ggcattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     3241 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     3301 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     3361 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     3421 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     3481 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     3541 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     3601 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     3661 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     3721 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     3781 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     3841 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     3901 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     3961 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     4021 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     4081 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     4141 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     4201 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     4261 tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa
     4321 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     4381 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     4441 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     4501 ggcgatggcc cacgctggct aagaactcat cagcctcccc ggtccatcta cccacgtacc
     4561 aatgcaccaa ttggccacaa tgacggctac tacatggtgc cattccttcc tctttatagg
     4621 aatggagact acctcctgtc caacaatgct cttggatacg agtacgccta cctgttggac
     4681 ccaggtcatt gcacaacacc agaaatgccc tctgatctgc aaaagacgtg aatatctgtt
     4741 cagacaccca tatccactct gttccacaca ggtcagaggt ttgtccagga gttcttgaca
     4801 gaggtgtaaa aagtactcaa aaattttact caagtgaaag tacaagtact tagggaaaat
     4861 tttactcaat taaaagtaaa agtatctggc tagaatctta cttgagtaaa agtaaaaaag
     4921 tactccatta aaattgtact tgagtattaa ggaagtaaaa gtaaaagcaa gaaagaaaac
     4981 tagagattct tgtttaagct tttaatctca aaaaacatta aatgaaatgc atacaaggtt
     5041 ttatcctgct ttagaactgt ttgtatttaa ttatcaaact ataagacaga caatctaatg
     5101 ccagtacacg ctactcaaag ttgtaaaacc tcagatttaa cttcagtaga agctgattct
     5161 caaaattgtt agtgtcaagc ctagctcttt tggggctgaa aagcaatcct gcagtgctga
     5221 aaagcctctc acaggcagcc gatgcgggaa gaggtgtatt agtcttgata gagaggctgc
     5281 aaatagcagg aaacgtgagc agagactccc tggtgtctga aacacaggcc catttacgcc
     5341 ttaagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     5401 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     5461 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt
     5521 ttttaaagca agtaaaacct ctacaaatgt ggtatggctg attatgatct agagtcgcgg
     5581 ccgctttact tgtacagctc gtccatgccg ccggtggagt ggtggccctc ggcgcgctcg
     5641 tactgttcca cgatggtgta gtcctcgttg tgggaggtga tgtccagctt gatgttgacg
     5701 ttgtaggcgc cgggcagctg cacgggcttc ttggccttgt aggtggtctt gacctcggcg
     5761 tcgtagtggc cgccgtcctt cagcttcagc ctctgcttga tctcgccctt cagggcgccg
     5821 tcctcggggt acatccgctc ggaggaggcc tcccagccca tggtcttctt ctgcattacg
     5881 gggccgtcgg aggggaagtt ggtgccgcgc agcttcacct tgtagatgaa ctcgccgtcc
     5941 tgcagggagg agtcctgggt cacggtcacc acgccgccgt cctcgaagtt catcacgcgc
     6001 tcccacttga agccctcggg gaaggacagc ttcaagtagt cggggatgtc ggcggggtgc
     6061 ttcacgtagg ccttggagcc gtacatgaac tgaggggaca ggatgtccca ggcgaagggc
     6121 agggggccgc ccttggtcac cttcagcttg gcggtctggg tgccctcgta ggggcggccc
     6181 tcgccctcgc cctcgatctc gaactcgtgg ccgttcacgg agccctccat gtgcaccttg
     6241 aagcgcatga actccttgat gatggccatg ttgtcatcct cgcccttgct caccatggtg
     6301 gcgaccggta atgtcagacc tggtaactcc ttactgtaac ttattgcacc tgatgaacaa
     6361 aagatgtgaa ctgaacgcaa gtaggaggct aatagaggct agtacagtac aaagatcccc
     6421 aaatgatgtg tgccggcctt agtggaggca gacccctggt tatatacctg tgctgcagaa
     6481 agaggggctt acaaatggat ctctctggaa tggcattatc tcatggtggg aacagtgttg
     6541 tcagcagaat cagcggggtg gactctgacc tgagaatgac agtctagcac tgaatggctc
     6601 agacaatact cttgtggcca actatcaccg gaaaataagt gcctgccaaa acaaaccaga
     6661 ccagatgaag ctacatggct gtagaacgtc agtatttaat gtgttgtggc cacttggttc
     6721 accagattta agagctaatt tttttaaata tcacagataa tcaatcgctt ttttttttct
     6781 tgactgaaaa gcttagactg gaaaattctt tgaaaaagtt tgcactacag aaaataaata
     6841 cataacatcc tgcactgaat gcacactatt aatagaaaag agagcattat tgtaggaaat
     6901 gtattgtata tgtacattct gtgcatttca caattttctc gagcaggaaa cagctatgac
     6961 catgattacg ccaagctatc aactttgtat agaaaagttg gtaatacgac tcactatagg
     7021 gcgaattggg taccgggccc cccctcgagg tcgacggtat cgataagctt gatatcgaat
     7081 tcctgcagcc cgggggatcc actagttcta gagcggccgc caccgcggtg gagctccagc
     7141 ttttgttccc tttagtgagg gttaattcaa gtttgt
//
