LOCUS       sequence-461800-        5185 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express H2B-GFP in zebrafish..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5185)
  TITLE     Screening of functional maternal-specific chromatin regulators in 
            early embryonic development of zebrafish
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5185)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pCS2-zH2B-GFP
FEATURES             Location/Qualifiers
     source          1..5185
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        40..58
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     40..57
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      469..478
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(475..477,478..480,481..1194)
                     /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(475..477,478..480,481..1191)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(520..541)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(781..800)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1128..1149
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1242..1376
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1266..1285)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1320..1339
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1489..1509)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1489..1507)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1528..1544)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1528..1544)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1541..1563)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1552..1568
                     /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(1576..1582,1583..1600,1601..1606))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1621..1642
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1759..1776)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1930..2518)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2010..2029)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2689..3480,3481..3549))
                     /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     3312..3331
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3550..3654)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3680..4135)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3817..3838)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4029..4048
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(4206..5185,1..5)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 ggaaccgcca ttctgcctgg ggacgtcgga gcaagcttga tttaggtgac actatagaat
       61 acaagctact tgttcttttt gcagatgcct gaaccagcaa aagccgcacc caagaagggc
      121 tccaaaaaag cggtgaccaa gaccgctgga aaaggaggaa agaagcgcaa gaggaccagg
      181 aaggagagct acgctatcta cgtctacaaa gtgttgaagc aagttcaccc agacaccggg
      241 atctcctcca aggcgatggg catcatgaac tcgttcgtca atgacatctt cgagcgcatc
      301 gccggtgagg cttctcgtct cgctcactac aacaagcgct ccaccatcac ttccagagag
      361 atccagaccg ccgtgcgtct gctgctgccc ggtgagctgg ccaaacacgc cgtgtctgag
      421 ggcaccaagg ctgtgaccaa gtacaccagc tccaaggatc caccggtcgc caccatggtg
      481 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
      541 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
      601 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
      661 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
      721 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
      781 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
      841 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
      901 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
      961 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     1021 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1081 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1141 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaagcggc
     1201 ccctctcgag cctctagaac tatagtgagt cgtattacgt agatccagac atgataagat
     1261 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     1321 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     1381 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaat
     1441 tcgcggccgc ggcgccaatg cattgggccc ggtacccagc ttttgttccc tttagtgagg
     1501 gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     1561 gctcacaatt ccacacaaca tacgagccgg gagcataaag tgtaaagcct ggggtgccta
     1621 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     1681 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     1741 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     1801 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     1861 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     1921 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     1981 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     2041 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     2101 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     2161 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     2221 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     2281 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     2341 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     2401 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     2461 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     2521 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     2581 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     2641 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     2701 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     2761 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     2821 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     2881 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     2941 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     3001 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     3061 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     3121 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     3181 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     3241 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     3301 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     3361 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     3421 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     3481 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     3541 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     3601 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     3661 tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     3721 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     3781 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     3841 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     3901 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     3961 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     4021 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     4081 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
     4141 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     4201 cgccagtcga ccgccaattc aatatggcgt atatggactc atgccaattc aatatggtgg
     4261 atctggacct gtgccaattc aatatggcgt atatggactc gtgccaattc aatatggtgg
     4321 atctggaccc cagccaattc aatatggcgg acttggcacc atgccaattc aatatggcgg
     4381 acctggcact gtgccaactg gggaggggtc tacttggcac ggtgccaagt ttgaggaggg
     4441 gtcttggccc tgtgccaagt ccgccatatt gaattggcat ggtgccaata atggcggcca
     4501 tattggctat atgccaggat caatatatag gcaatatcca atatggccct atgccaatat
     4561 ggctattggc caggttcaat actatgtatt ggccctatgc catatagtat tccatatatg
     4621 ggttttccta ttgacgtaga tagcccctcc caatgggcgg tcccatatac catatatggg
     4681 gcttcctaat accgcccata gccactcccc cattgacgtc aatggtctct atatatggtc
     4741 tttcctattg acgtcatatg ggcggtccta ttgacgtata tggcgcctcc cccattgacg
     4801 tcaattacgg taaatggccc gcctggctca atgcccattg acgtcaatag gaccacccac
     4861 cattgacgtc aatgggatgg ctcattgccc attcatatcc gttctcacgc cccctattga
     4921 cgtcaatgac ggtaaatggc ccacttggca gtacatcaat atctattaat agtaacttgg
     4981 caagtacatt actattggaa gtacgccagg gtacattggc agtactccca ttgacgtcaa
     5041 tggcggtaaa tggcccgcga tggctgccaa gtacatcccc attgacgtca atggggaggg
     5101 gcaatgacgc aaatgggcgt tccattgacg taaatgggcg gtaggcgtgc ctaatgggag
     5161 gtctatataa gcaatgctcg tttag
//
