LOCUS       Exported                7862 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  contains clec14a enhancer sequence, and clec14a specific gene 
            promoter driving expression of eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    pT2-cryR;clecl14aCE1-P1Egfp
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7862)
  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 7862)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7862
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     629..645
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    665..685
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    1193..1217
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1870..1879
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1876..2592
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             1876..2592
                     /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(1921..1942)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2182..2201)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2529..2550
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2593..2658
                     /label=MCS
                     /note="multiple cloning site"
     protein_bind    complement(2679..2703)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2729..2749)
                     /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(2757..2773)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2789..2923
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3595..3611)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3595..3611)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3648..3668)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3648..3666)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3687..3703)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3700..3722)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3711..3727
                     /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(3735..3765)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3780..3801
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3918..3935)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4089..4677)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4169..4188)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4848..5708)
                     /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     5471..5490
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5709..5813)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5976..5997)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    6889..7010
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(7133..7843)
                     /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(7213..7236)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(7411..7430)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     7680..7698
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      7840..7849
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
ORIGIN
        1 agacctggta actccttact gtaacttatt gcacctgatg aacaaaagat gtgaactgaa
       61 cgcaagtagg aggctaatag aggctagtac agtacaaaga tccccaaatg atgtgtgccg
      121 gccttagtgg aggcagaccc ctggttatat acctgtgctg cagaaagagg ggcttacaaa
      181 tggatctctc tggaatggca ttatctcatg gtgggaacag tgttgtcagc agaatcagcg
      241 gggtggactc tgacctgaga atgacagtct agcactgaat ggctcagaca atactcttgt
      301 ggccaactat caccggaaaa taagtgcctg ccaaaacaaa ccagaccaga tgaagctaca
      361 tggctgtaga acgtcagtat ttaatgtgtt gtggccactt ggttcaccag atttaagagc
      421 taattttttt aaatatcaca gataatcaat cgcttttttt tttcttgact gaaaagctta
      481 gactggaaaa ttctttgaaa aagtttgcac tacagaaaat aaatacataa catcctgcac
      541 tgaatgcaca ctattaatag aaaagagagc attattgtag gaaatgtatt gtatatgtac
      601 attctgtgca tttcacaatt ttctcgagca ggaaacagct atgaccatga ttacgccaag
      661 ctatcaactt tgtatagaaa agttgtcatt gttgtttaaa aaagcagcga gctctcgtaa
      721 taaaacacct gtatcacctt catttcaact ttccttcttt gttctttcta ggcatacttg
      781 tagctccttt tcctgttacc cataattcca tttcctgtcc ttcttcccca cccccctggc
      841 tctctagact gcagaacaga gagtcgagtg ctcctgcaac ctcacttcct gccattcaga
      901 gccgtttcct ccaggaagca tctgctgctc tctcatgaag cggcctgttg gaggaacaga
      961 ataataatac ggtgttagtc atgggacatt caacaccaca ttagtcccat gtgagctctt
     1021 gaaaacacac gcggaggtca atcaacacgc atttgaaact gacagcgttg agtgcgacat
     1081 gtaaacacag aggtatagta ctactgtaaa gaacaatccc tgatgtaatg agcaacgctt
     1141 cctggtattt ttcccagagt tgttttgtta cagacacaat gaacaagtca aagcaagttt
     1201 gtacaaaaaa gcaggctcgc tagttgactt gacatatatg cacgtaatgt gttctgtaaa
     1261 tatacaataa tttattttcc acttaaagac ccttaaaaat agggcactga tgtaaagtac
     1321 atttttaaat ggcgaggtat aaagtggaat aattttcaaa actttatact cactactctt
     1381 gaatattttt agaagtgcta ctttttactc atattttgag tagcaggtac ttttacttgc
     1441 actacatatt tttgggaagt aaagatactt ttacttgagc atgatttttc agtcctcttt
     1501 ccaccactgg ctacgatgaa acctgaaacc ttcaaggaga tgatgggtgt ggatgcatga
     1561 acggtttatc agccctatgc caagaaacac aggtttcctc gtcatttaaa gctcatccgt
     1621 cactctgctg tgggagtttg gcagtgggat tgtgtattat ggaagcttca gtttcaccag
     1681 gaacagattt ttgttcatag ttgcgattgt tttgcttcgg cagcaccacc acccaagagg
     1741 aagcgaatgg ctgtatcaag gctgaacaga cttcctgtgc ttgttgtctt gggaggacct
     1801 cacagggacg gctaagaaag ccagggggag aaaaagcaga caatatcatt ttagtttgag
     1861 tctgcgatcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     1921 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     1981 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     2041 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     2101 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     2161 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     2221 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     2281 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     2341 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     2401 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     2461 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     2521 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     2581 gagctgtaca agtccggact cagatctcga gctcaagctt cgaattctgc agtcgacggt
     2641 accgcgggcc cgggatccac cggatctaga taactgatac ccagctttct tgtacaaagt
     2701 ggggcgcgcc tacgtaacta gtctcgagca actttattat acatagttga taattcactg
     2761 gccgtcgttt tacggtacca tcgatgatga tccagacatg ataagataca ttgatgagtt
     2821 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     2881 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     2941 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct
     3001 ctacaaatgt ggtatggctg attatgatcc tctagatcag atctgcgaag atacggccac
     3061 gggtgctctt gatcctgtgg ctgattttgg actgtgctgc tcgcagctgc tgatgaatca
     3121 catacttcct ccattttctt ccactgattg actgttataa tttccctaat ttccaggtca
     3181 aggtgctgtg cattgtggta atagatgtga catgacgtca cttccaaagg accaatgaac
     3241 atgtctgacc aatttcatat aatgtgaaaa cgattttcat aggcagaata aataacattt
     3301 aaattaaact gggcatcagc gcaattcaat tggtttggta atagcaaggg aaaatagaat
     3361 gaagtgatct ccaaaaaata agtacttttt gactgtaaat aaaattgtaa ggagtaaaaa
     3421 gtactttttt ttctaaaaaa atgtaattaa gtaaaagtaa aagtattgat ttttaattgt
     3481 actcaagtaa agtaaaaatc cccaaaaata atacttaagt acagtaatca agtaaaatta
     3541 ctcaagtact ttacacctct ggttcttgac cccctacctt cagcaagccc agcagatcca
     3601 ctagttctag agcggccgcc accgcggtgg agctccagct tttgttccct ttagtgaggg
     3661 ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     3721 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3781 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3841 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3901 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3961 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4021 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4081 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4141 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4201 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4261 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4321 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4381 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4441 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4501 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     4561 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4621 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4681 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggc
     4741 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4801 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4861 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4921 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4981 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5041 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5101 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5161 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5221 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5281 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5341 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5401 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5461 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5521 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5581 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5641 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5701 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5761 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5821 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     5881 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     5941 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     6001 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     6061 gctggctaag aactcatcag cctccccggt ccatctaccc acgtaccaat gcaccaattg
     6121 gccacaatga cggctactac atggtgccat tccttcctct ttataggaat ggagactacc
     6181 tcctgtccaa caatgctctt ggatacgagt acgcctacct gttggaccca ggtcattgca
     6241 caacaccaga aatgccctct gatctgcaaa agacgtgaat atctgttcag acacccatat
     6301 ccactctgtt ccacacaggt cagaggtttg tccaggagtt cttgacagag gtgtaaaaag
     6361 tactcaaaaa ttttactcaa gtgaaagtac aagtacttag ggaaaatttt actcaattaa
     6421 aagtaaaagt atctggctag aatcttactt gagtaaaagt aaaaaagtac tccattaaaa
     6481 ttgtacttga gtattaagga agtaaaagta aaagcaagaa agaaaactag agattcttgt
     6541 ttaagctttt aatctcaaaa aacattaaat gaaatgcata caaggtttta tcctgcttta
     6601 gaactgtttg tatttaatta tcaaactata agacagacaa tctaatgcca gtacacgcta
     6661 ctcaaagttg taaaacctca gatttaactt cagtagaagc tgattctcaa aattgttagt
     6721 gtcaagccta gctcttttgg ggctgaaaag caatcctgca gtgctgaaaa gcctctcaca
     6781 ggcagccgat gcgggaagag gtgtattagt cttgatagag aggctgcaaa tagcaggaaa
     6841 cgtgagcaga gactccctgg tgtctgaaac acaggcccat ttacgcctta agatacattg
     6901 atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt
     6961 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca
     7021 attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt
     7081 aaaacctcta caaatgtggt atggctgatt atgatctaga gtcgcggccg ctttacttgt
     7141 acagctcgtc catgccgccg gtggagtggt ggccctcggc gcgctcgtac tgttccacga
     7201 tggtgtagtc ctcgttgtgg gaggtgatgt ccagcttgat gttgacgttg taggcgccgg
     7261 gcagctgcac gggcttcttg gccttgtagg tggtcttgac ctcggcgtcg tagtggccgc
     7321 cgtccttcag cttcagcctc tgcttgatct cgcccttcag ggcgccgtcc tcggggtaca
     7381 tccgctcgga ggaggcctcc cagcccatgg tcttcttctg cattacgggg ccgtcggagg
     7441 ggaagttggt gccgcgcagc ttcaccttgt agatgaactc gccgtcctgc agggaggagt
     7501 cctgggtcac ggtcaccacg ccgccgtcct cgaagttcat cacgcgctcc cacttgaagc
     7561 cctcggggaa ggacagcttc aagtagtcgg ggatgtcggc ggggtgcttc acgtaggcct
     7621 tggagccgta catgaactga ggggacagga tgtcccaggc gaagggcagg gggccgccct
     7681 tggtcacctt cagcttggcg gtctgggtgc cctcgtaggg gcggccctcg ccctcgccct
     7741 cgatctcgaa ctcgtggccg ttcacggagc cctccatgtg caccttgaag cgcatgaact
     7801 ccttgatgat ggccatgttg tcatcctcgc ccttgctcac catggtggcg accggtaatg
     7861 tc
//
