LOCUS       sequence-326981-        4814 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reporter plasmid for the DHS-4D Otx2 enhancer element..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4814)
  AUTHORS   Kaufman ML, Goodson NB, Park KU, Schwanke M, Office E, Schneider SR,
            Abraham J, Hensley A, Jones KL, Brzezinski JA
  TITLE     Initiation of Otx2 expression in the developing mouse retina 
            requires a unique enhancer and either Ascl1 or Neurog2 activity.
  JOURNAL   Development. 2021 Jun 15;148(12). pii: 269198. doi: 
            10.1242/dev.199399. Epub 2021 Jun 18.
  PUBMED    34143204
REFERENCE   2  (bases 1 to 4814)
  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"; journalName: 
            "Development."; date: "2021-06-15"; volume: "148(12). pii: 269198. 
            doi"; pages: " 10.1242/dev.199399. Epub 2021 Jun 18"
COMMENT     Sequence Label: pMin-DHS-4D-GFP
FEATURES             Location/Qualifiers
     source          1..4814
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             274..282
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     promoter        605..635
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     regulatory      647..656
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(653..655,656..658,659..1369)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(653..655,656..658,659..1369)
                     /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(698..719)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(959..978)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1306..1327
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1397..1417
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1421..1441
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1445..1465
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1608..1729
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1645..1664)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1699..1718
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1736..2191)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1873..1894)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2085..2104
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2218..2322
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2324..2681
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2324..2515
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2347..2367)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2532..2667
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2716..3510
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2716..3510
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2770..2789)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3380..3399
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3698..3717)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3742..3789
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4118..4706
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4607..4626
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atgcattagt tattactagc gctaccggac tcagatctcg agctcaagct tcgaattcga
       61 cctccacccc tcacctcttc tctcttctcc ccttgtttgg ctactggctc agaggctgca
      121 gtgcagaccc tgttgctctg gtggcacgtg cagtgtacac ggcatatggc tggctggtcg
      181 tacacagaag aggataactg aagccaaaca gacccagcgc tgttattaca gcagatgggg
      241 ggaagcgtgg cattcaatat ttagacagat gccacctcct tattccacct gtctggaagt
      301 tggacattat acagaacgag tgtctgtcta atgaggaagg aaacaaaggg cgcgtttatg
      361 aagctcggca gagcggcgtg tttgctgagc aggcacaata taattgttga ccataacaat
      421 aaacgtgaac tgcttgctac tggtcggaac cgctgataca ggagcacaat tacagtagga
      481 gcgaggggtg gctggttttg ccacctgctt tggtctttaa tttgggtgaa tgaacactag
      541 atgggactcg gtgtaggaga cgcaagcctg gaagaaggta ccgcgggccc gggatcggcg
      601 cgccagaggg tatataatgg aagctcgact tccagatcca ccggtcgcca ccatggtgag
      661 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
      721 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
      781 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
      841 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
      901 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
      961 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tagtgaaccg
     1021 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     1081 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     1141 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     1201 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     1261 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     1321 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagc ggccgcgact
     1381 cagatctcga gctgatccaa aaaagaagag aaaggtagat ccaaaaaaga agagaaaggt
     1441 agatccaaaa aagaagagaa aggtaggatc caccggatct agataagcgg ccgcgactct
     1501 agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     1561 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     1621 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     1681 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt
     1741 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     1801 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     1861 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     1921 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     1981 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     2041 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     2101 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     2161 gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc
     2221 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     2281 taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc
     2341 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     2401 gtatgcaaag catgcatctc aattaatcag caaccaggtg tggaaagtcc ccaggctccc
     2461 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     2521 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     2581 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     2641 agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga
     2701 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     2761 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     2821 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     2881 gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     2941 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3001 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3061 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3121 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     3181 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     3241 gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     3301 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     3361 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     3421 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     3481 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     3541 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     3601 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     3661 tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac
     3721 cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt
     3781 cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga
     3841 gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt
     3901 cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag
     3961 gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4021 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4081 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4141 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4201 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4261 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4321 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4381 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4441 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4501 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4561 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4621 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4681 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4741 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4801 accgtattac cgcc
//
