LOCUS       sequence-268886-        5257 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of H2B-GFP mRNA for microinjection to visualize Histone 
            2B protein dynamics in S. purpuratus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5257)
  AUTHORS   Uchida A, Yajima M
  TITLE     An optogenetic approach to control protein localization during 
            embryogenesis of the sea urchin.
  JOURNAL   Dev Biol. 2018 Sep 1;441(1):19-30. doi: 10.1016/j.ydbio.2018.06.015.
            Epub 2018 Jun 26.
  PUBMED    29958898
REFERENCE   2  (bases 1 to 5257)
  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.ydbio.2018.06.015"; journalName: "Dev Biol"; date: 
            "2018-09-1- 1"; volume: "441"; issue: "1"; pages: "19-30"
COMMENT     Sequence Label: pSP64-H2B-GFP
FEATURES             Location/Qualifiers
     source          1..5257
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        814..832
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     814..831
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             937..1314
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      1327..1336
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1333..1335,1336..1338,1339..2052)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1333..1335,1336..1338,1339..2049)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1378..1399)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1639..1658)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1986..2007
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2088..2222
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2112..2131)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2166..2185
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2335..2355)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2335..2353)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2374..2390)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2374..2390)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2387..2409)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2398..2414
                     /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(2422..2428,2429..2446,2447..2452))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2467..2488
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2605..2622)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2776..3364)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2856..2875)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3535..4326,4327..4395))
                     /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     4158..4177
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4396..4500)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4526..4981)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4663..4684)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4875..4894
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(5052..5257,1..779)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 acttggcacg gtgccaagtt tgaggagggg tcttggccct gtgccaagtc cgccatattg
       61 aattggcatg gtgccaataa tggcggccat attggctata tgccaggatc aatatatagg
      121 caatatccaa tatggcccta tgccaatatg gctattggcc aggttcaata ctatgtattg
      181 gccctatgcc atatagtatt ccatatatgg gttttcctat tgacgtagat agcccctccc
      241 aatgggcggt cccatatacc atatatgggg cttcctaata ccgcccatag ccactccccc
      301 attgacgtca atggtctcta tatatggtct ttcctattga cgtcatatgg gcggtcctat
      361 tgacgtatat ggcgcctccc ccattgacgt caattacggt aaatggcccg cctggctcaa
      421 tgcccattga cgtcaatagg accacccacc attgacgtca atgggatggc tcattgccca
      481 ttcatatccg ttctcacgcc ccctattgac gtcaatgacg gtaaatggcc cacttggcag
      541 tacatcaata tctattaata gtaacttggc aagtacatta ctattggaag tacgccaggg
      601 tacattggca gtactcccat tgacgtcaat ggcggtaaat ggcccgcgat ggctgccaag
      661 tacatcccca ttgacgtcaa tggggagggg caatgacgca aatgggcgtt ccattgacgt
      721 aaatgggcgg taggcgtgcc taatgggagg tctatataag caatgctcgt ttagggaacc
      781 gccattctgc ctggggacgt cggagcaagc ttgatttagg tgacactata gaatacaagc
      841 tacttgttct ttttgcagga tcccatcgat tcgaattcaa ggcctctcga gcctctagaa
      901 ctatagtgag tcgtattact cgacggtacc gccaccatgc cagagccagc gaagtctgct
      961 cccgccccga aaaagggctc caagaaggcg gtgactaagg cgcagaagaa aggcggcaag
     1021 aagcgcaagc gcagccgcaa ggagagctat tccatctatg tgtacaaggt tctgaagcag
     1081 gtccaccctg acaccggcat ttcgtccaag gccatgggca tcatgaattc gtttgtgaac
     1141 gacattttcg agcgcatcgc aggtgaggct tcccgcctgg cgcattacaa caagcgctcg
     1201 accatcacct ccagggagat ccagacggcc gtgcgcctgc tgctgcctgg ggagttggcc
     1261 aagcacgccg tgtccgaggg tactaaggcc atcaccaagt acaccagcgc taaggatcca
     1321 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     1381 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     1441 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     1501 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     1561 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     1621 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     1681 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1741 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1801 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1861 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1921 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1981 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     2041 ctgtacaagt aaagcggccc tagaactata gtgagtcgta ttacgtagat ccagacatga
     2101 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
     2161 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
     2221 ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt
     2281 tttaattcgc ggccgcggcg ccaatgcatt gggcccggta cccagctttt gttcccttta
     2341 gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     2401 ttatccgctc acaattccac acaacatacg agccgggagc ataaagtgta aagcctgggg
     2461 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     2521 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     2581 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     2641 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     2701 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     2761 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     2821 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     2881 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     2941 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     3001 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     3061 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3121 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     3181 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     3241 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     3301 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     3361 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     3421 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     3481 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     3541 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     3601 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     3661 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     3721 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     3781 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     3841 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     3901 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     3961 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     4021 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     4081 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     4141 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     4201 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     4261 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     4321 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     4381 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     4441 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     4501 cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg
     4561 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     4621 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     4681 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     4741 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     4801 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     4861 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     4921 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     4981 tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     5041 ctattacgcc agtcgaccgc caattcaata tggcgtatat ggactcatgc caattcaata
     5101 tggtggatct ggacctgtgc caattcaata tggcgtatat ggactcgtgc caattcaata
     5161 tggtggatct ggaccccagc caattcaata tggcggactt ggcaccatgc caattcaata
     5221 tggcggacct ggcactgtgc caactgggga ggggtct
//
