LOCUS       Exported                7476 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  contains lmo2 enhancer sequence and a basal promoter driving 
            expression of eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    pT2-cryR;lmo2CE1-basEgfp
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7476)
  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 7476)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7476
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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    1154..1178
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1255..1264
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1261..1977
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             1261..1977
                     /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(1306..1327)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1567..1586)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1914..1935
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1978..2043
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2167..2288
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(2295..2319)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2345..2365)
                     /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(2373..2389)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2405..2539
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3211..3227)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3211..3227)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3264..3284)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3264..3282)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3303..3319)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3316..3338)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3327..3343
                     /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(3351..3381)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3396..3417
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3534..3551)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3705..4293)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3785..3804)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4464..5324)
                     /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     5087..5106
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5325..5429)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5592..5613)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    6505..6626
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6749..7459)
                     /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(6829..6852)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(7027..7046)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     7296..7314
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      7456..7465
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
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 aaagttgcaa aaagtcatca tggccaacag aatgttatgt
      721 ggaagaatgt gatctaatgt caaagaaaat gattctcgtg acacatcggg agtattgcat
      781 gacaatgacc agaaaaaaaa acgctaataa cagagatgtc ataagccatg ttgctgtaca
      841 gcatttacca cacccctaac cctttttgaa gatttatgag ttaatggttt tcctgcagta
      901 gatgagttga cggtttccaa ggctctgttt cagaagccat ccttctatta aaggagcctt
      961 cagccaatca cagtccagca gggcaatgaa gtgaagcgaa caggaagtca acacgactct
     1021 tacaccccct gccagggttc atctgtgctc atttcctgca catcctctgg ctttgctgtg
     1081 tccaatccac aggaaacggt ggacgcctta tgcacacagt ttaacattta tttaaaacaa
     1141 actgtagaag caaacaagtt tgtacaaaaa agcaggctag actctagagg gtatataatg
     1201 gatctcgcgt ctcagcctca ctttgagctc ctccacacgc tagcgctacc ggtcgccacc
     1261 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     1321 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     1381 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1441 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     1501 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     1561 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1621 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1681 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     1741 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     1801 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1861 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1921 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc
     1981 ggactcagat ctcgagctca agcttcgaat tctgcagtcg acggtaccgc gggcccggga
     2041 tccaccggat ctagataact gatcataatc agccatacca catttgtaga ggttttactt
     2101 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     2161 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     2221 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     2281 gtatcttaac gcgtacccag ctttcttgta caaagtgggg cgcgcctacg taactagtct
     2341 cgagcaactt tattatacat agttgataat tcactggccg tcgttttacg gtaccatcga
     2401 tgatgatcca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     2461 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     2521 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     2581 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta tggctgatta
     2641 tgatcctcta gatcagatct gcgaagatac ggccacgggt gctcttgatc ctgtggctga
     2701 ttttggactg tgctgctcgc agctgctgat gaatcacata cttcctccat tttcttccac
     2761 tgattgactg ttataatttc cctaatttcc aggtcaaggt gctgtgcatt gtggtaatag
     2821 atgtgacatg acgtcacttc caaaggacca atgaacatgt ctgaccaatt tcatataatg
     2881 tgaaaacgat tttcataggc agaataaata acatttaaat taaactgggc atcagcgcaa
     2941 ttcaattggt ttggtaatag caagggaaaa tagaatgaag tgatctccaa aaaataagta
     3001 ctttttgact gtaaataaaa ttgtaaggag taaaaagtac ttttttttct aaaaaaatgt
     3061 aattaagtaa aagtaaaagt attgattttt aattgtactc aagtaaagta aaaatcccca
     3121 aaaataatac ttaagtacag taatcaagta aaattactca agtactttac acctctggtt
     3181 cttgaccccc taccttcagc aagcccagca gatccactag ttctagagcg gccgccaccg
     3241 cggtggagct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
     3301 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     3361 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     3421 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     3481 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     3541 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3601 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3661 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3721 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3781 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3841 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     3901 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     3961 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4021 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4081 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4141 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4201 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4261 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4321 tctgacgctc agtggaacga aaactcacgt taaggcattt tggtcatgag attatcaaaa
     4381 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     4441 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     4501 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     4561 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     4621 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4681 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     4741 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     4801 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     4861 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     4921 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     4981 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5041 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     5101 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5161 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5221 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     5281 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     5341 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     5401 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg
     5461 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     5521 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     5581 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     5641 aagggcgaaa aaccgtctat cagggcgatg gcccacgctg gctaagaact catcagcctc
     5701 cccggtccat ctacccacgt accaatgcac caattggcca caatgacggc tactacatgg
     5761 tgccattcct tcctctttat aggaatggag actacctcct gtccaacaat gctcttggat
     5821 acgagtacgc ctacctgttg gacccaggtc attgcacaac accagaaatg ccctctgatc
     5881 tgcaaaagac gtgaatatct gttcagacac ccatatccac tctgttccac acaggtcaga
     5941 ggtttgtcca ggagttcttg acagaggtgt aaaaagtact caaaaatttt actcaagtga
     6001 aagtacaagt acttagggaa aattttactc aattaaaagt aaaagtatct ggctagaatc
     6061 ttacttgagt aaaagtaaaa aagtactcca ttaaaattgt acttgagtat taaggaagta
     6121 aaagtaaaag caagaaagaa aactagagat tcttgtttaa gcttttaatc tcaaaaaaca
     6181 ttaaatgaaa tgcatacaag gttttatcct gctttagaac tgtttgtatt taattatcaa
     6241 actataagac agacaatcta atgccagtac acgctactca aagttgtaaa acctcagatt
     6301 taacttcagt agaagctgat tctcaaaatt gttagtgtca agcctagctc ttttggggct
     6361 gaaaagcaat cctgcagtgc tgaaaagcct ctcacaggca gccgatgcgg gaagaggtgt
     6421 attagtcttg atagagaggc tgcaaatagc aggaaacgtg agcagagact ccctggtgtc
     6481 tgaaacacag gcccatttac gccttaagat acattgatga gtttggacaa accacaacta
     6541 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     6601 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     6661 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg
     6721 ctgattatga tctagagtcg cggccgcttt acttgtacag ctcgtccatg ccgccggtgg
     6781 agtggtggcc ctcggcgcgc tcgtactgtt ccacgatggt gtagtcctcg ttgtgggagg
     6841 tgatgtccag cttgatgttg acgttgtagg cgccgggcag ctgcacgggc ttcttggcct
     6901 tgtaggtggt cttgacctcg gcgtcgtagt ggccgccgtc cttcagcttc agcctctgct
     6961 tgatctcgcc cttcagggcg ccgtcctcgg ggtacatccg ctcggaggag gcctcccagc
     7021 ccatggtctt cttctgcatt acggggccgt cggaggggaa gttggtgccg cgcagcttca
     7081 ccttgtagat gaactcgccg tcctgcaggg aggagtcctg ggtcacggtc accacgccgc
     7141 cgtcctcgaa gttcatcacg cgctcccact tgaagccctc ggggaaggac agcttcaagt
     7201 agtcggggat gtcggcgggg tgcttcacgt aggccttgga gccgtacatg aactgagggg
     7261 acaggatgtc ccaggcgaag ggcagggggc cgcccttggt caccttcagc ttggcggtct
     7321 gggtgccctc gtaggggcgg ccctcgccct cgccctcgat ctcgaactcg tggccgttca
     7381 cggagccctc catgtgcacc ttgaagcgca tgaactcctt gatgatggcc atgttgtcat
     7441 cctcgccctt gctcaccatg gtggcgaccg gtaatg
//
