LOCUS       sequence-263531-        4775 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gata2a endothelial enhancer (x6) and basal promoter driving egfp in 
            pTol1 backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4775)
  AUTHORS   Shin M, Nozaki T, Idrizi F, Isogai S, Ogasawara K, Ishida K, Yuge S,
            Roscoe B, Wolfe SA, Fukuhara S, Mochizuki N, Deguchi T, Lawson ND
  TITLE     Valves Are a Conserved Feature of the Zebrafish Lymphatic System.
  JOURNAL   Dev Cell. 2019 Nov 4;51(3):374-386.e5. doi: 
            10.1016/j.devcel.2019.08.019. Epub 2019 Sep 26.
  PUBMED    31564611
REFERENCE   2  (bases 1 to 4775)
  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.devcel.2019.08.019"; journalName: "Dev Cell"; date: 
            "2019-11-4- 4"; volume: "51"; issue: "3"; pages: "374-386.e5"
COMMENT     Sequence Label: pTone-gata2aECE-EGFP
FEATURES             Location/Qualifiers
     source          1..4775
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    628..648
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    934..958
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1035..1044
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1041..1043,1044..1046,1047..1757)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1041..1043,1044..1046,1047..1757)
                     /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(1086..1107)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1347..1366)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1694..1715
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1758..1823
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    1947..2068
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(2075..2099)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2125..2145)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    2167..2301
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2502..2518)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2554..2570)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2567..2589)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2578..2594
                     /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(2602..2608,2609..2626,2627..2632))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2647..2668
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2785..2802)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2956..3544)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3036..3055)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3715..4506,4507..4575))
                     /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     4338..4357
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4576..4680)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4748..4766
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ccctttagcc agtagcggtt
      421 ctaggcacgg gccgtccggg cggtggcctg gggcggaaaa ctgaaggggg gcggcaccgg
      481 cggctcagcc ctttgtaata tattaatatg caccactatt ggtttactta tgtcacagtt
      541 tgtaagtttg taacagcctg aacctggccg cgcgccattg ggatggaacc ctcgaggggc
      601 ccgctagcga tatccctgca gggctatcaa ctttgtatag aaaagttgag ttgtttccta
      661 tccggatatc tgcccgggcc cgataaagtt gtttcctatc cggatatctg cccgggcccg
      721 ataacacgtc acgcacgtgc agtactagtt gtttcctatc cggatatctg cccgggcccg
      781 ataaagttgt ttcctatccg gatatctgcc cgggcccgat aacacgtcac gcacgtgcag
      841 tactagttgt ttcctatccg gatatctgcc cgggcccgat aaagttgttt cctatccgga
      901 tatctgcccg ggcccgataa gcttaattct cgagcaagtt tgtacaaaaa agcaggctag
      961 actctagagg gtatataatg gatctcgcgt ctcagcctca ctttgagctc ctccacacgc
     1021 tagcgctacc ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc
     1081 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     1141 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     1201 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     1261 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     1321 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     1381 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     1441 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     1501 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     1561 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     1621 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     1681 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     1741 tggacgagct gtacaagtcc ggactcagat ctcgagctca agcttcgaat tctgcagtcg
     1801 acggtaccgc gggcccggga tccaccggat ctagataact gatcataatc agccatacca
     1861 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     1921 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     1981 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     2041 gtttgtccaa actcatcaat gtatcttaac gcgtacccag ctttcttgta caaagtgggg
     2101 cgcgcctacg taactagtct cgagcaactt tattatacat agttgataag gcgcgccagg
     2161 cctgtagatc cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     2221 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     2281 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag
     2341 ggggaggtgt gggaggtttt ttaattaaaa catttcagtt gacgaagaca aacaaagttc
     2401 tgttgtgact atgggggggg ggggcgcctg gggatggtct cgcccgggga gtaattcagg
     2461 gtagaaccgc cactggctaa aggaagcttg gcgtaatcat ggtcatagct gtttcctgta
     2521 tcccaatggc gcgccgagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     2581 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     2641 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     2701 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     2761 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     2821 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     2881 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     2941 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3001 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3061 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     3121 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     3181 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     3241 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3301 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     3361 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     3421 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     3481 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     3541 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     3601 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     3661 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     3721 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     3781 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     3841 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     3901 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     3961 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     4021 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     4081 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     4141 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     4201 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     4261 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     4321 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     4381 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     4441 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     4501 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     4561 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     4621 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     4681 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     4741 ctataaaaat aggcgtatca cgaggccctt tcgtc
//
