LOCUS       sequence-128564-        6046 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses zebrafish Chk1 mutant with C-term GFP fluorescent tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6046)
  AUTHORS   Zhang M, Kothari P, Mullins M, Lampson MA
  TITLE     Regulation of zygotic genome activation and DNA damage checkpoint 
            acquisition at the mid-blastula transition.
  JOURNAL   Cell Cycle. 2014;13(24):3828-38. doi: 10.4161/15384101.2014.967066.
  PUBMED    25558827
REFERENCE   2  (bases 1 to 6046)
  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: "Cell 
            Cycle."; date: "2014"; volume: "13(24):3828-38. doi"; pages: " 
            10.4161/15384101.2014.967066"
COMMENT     Sequence Label: pMZ031
FEATURES             Location/Qualifiers
     source          1..6046
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             join(1342..1344,1345..1347,1348..2061)
                     /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(1342..1344,1345..1347,1348..2058)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1387..1408)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1648..1667)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1995..2016
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2076..2095)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2077..2095)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    2100..2234
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2124..2143)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2178..2197
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2347..2367)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2347..2365)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2386..2402)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2386..2402)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2399..2421)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2410..2426
                     /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(2434..2440,2441..2458,2459..2464))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2479..2500
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2617..2634)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2788..3376)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2868..2887)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3547..4338,4339..4407))
                     /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     4170..4189
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4408..4512)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4538..4993)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4675..4696)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4887..4906
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atcccatcga ttcgaattca ggaccatggc tgtgcctttt
      121 gttaaagact gggatgtggt acaaactctt ggagagggag catatggaga ggtgcgactg
      181 ctggtcaaca agaaaacaga agaggctgtg gcggtgaaag ttgtggacat ggcaaaagcc
      241 aaggattgca tcgagaatgt gaagaaggag gtctgcatat gcaagatgct ttcacacccc
      301 aacattgtac gtttctttgg gcacagtgtt gggattacac atcgtgacat aaagcctgag
      361 aatattcttc ttgatgataa agataatctg aagatctctg actttggcct ggctaccatg
      421 ttcaggcacc gtggccgtga gcgagctttg aaccgtctgt gtggtactct gccctatgtt
      481 gccccagagt tgatgtcacg ctcatctttt aacgctcagc ctgcggacac ttgggcttgt
      541 ggcattgtgc tcactgcaat gttagctgga gagttaccct gggatcagcc gagtgaaaac
      601 tgtcaggaat atttggactg gctggaaaga aagacctacc ttacaccctg gaagaaaatt
      661 gatgcggtac cccttagtct gttgtctaag atattactgc acaatccaga agacaggttc
      721 accattcctg aaattaagaa acaccgctgg tttagcagaa gtttcaaatc agcagtacaa
      781 cgtcagggca tcacaccagc agccaagatt caacgggctg actctgagct gagacgaaaa
      841 aacagtgatg accgagcgca gatctccagc gaacagcctg agccacaggg attatgggag
      901 gagaaggagg ttacagttca caccgatgtc agtgtcagct ttgaacaacc gacctgtcct
      961 gaccacatgc tgctgggcga acaactactt ggcacaccgg gtgcagaaca gagcccctgg
     1021 cagcgtttgg tgaggagaat gaccaggttc tttactacag aaaatacaga ggcatcttgc
     1081 attgctctcc gtgattcttg tattgctttg ggtcacacat ggaaacagag ctgtaataat
     1141 caggcgacag tgtctacaat ggatcgtcgc aataataagc tgatattcat agtgcacttc
     1201 ctggaaatgg aggaacgtat tttagttgat ttccggctgt caaaggggga cggtttggaa
     1261 ttcaagcgaa tattcctgaa actcaaacag aagctgtctg acattattag caaccaaaag
     1321 gttttgccat tgattctcga gatggtgagc aagggcgagg agctgttcac cggggtggtg
     1381 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     1441 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     1501 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
     1561 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     1621 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     1681 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     1741 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     1801 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     1861 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     1921 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     1981 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     2041 atggacgagc tgtacaagta atctagattc tgcagcccta tagtgagtcg tattacgtag
     2101 atccagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
     2161 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     2221 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     2281 tgtgggaggt tttttaattc gcggccgcgg cgccaatgca ttgggcccgg tacccagctt
     2341 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     2401 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     2461 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     2521 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     2581 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     2641 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     2701 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     2761 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     2821 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     2881 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     2941 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3001 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3061 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3121 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     3181 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     3241 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     3301 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     3361 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     3421 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     3481 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     3541 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     3601 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     3661 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     3721 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     3781 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     3841 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     3901 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     3961 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4021 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4081 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4141 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     4201 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     4261 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     4321 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     4381 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     4441 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     4501 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg
     4561 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     4621 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     4681 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     4741 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     4801 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     4861 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     4921 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     4981 ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     5041 cgggcctctt cgctattacg ccagtcgacc atagccaatt caatatggcg tatatggact
     5101 catgccaatt caatatggtg gatctggacc tgtgccaatt caatatggcg tatatggact
     5161 cgtgccaatt caatatggtg gatctggacc ccagccaatt caatatggcg gacttggcac
     5221 catgccaatt caatatggcg gacttggcac tgtgccaact ggggaggggt ctacttggca
     5281 cggtgccaag tttgaggagg ggtcttggcc ctgtgccaag tccgccatat tgaattggca
     5341 tggtgccaat aatggcggcc atattggcta tatgccagga tcaatatata ggcaatatcc
     5401 aatatggccc tatgccaata tggctattgg ccaggttcaa tactatgtat tggccctatg
     5461 ccatatagta ttccatatat gggttttcct attgacgtag atagcccctc ccaatgggcg
     5521 gtcccatata ccatatatgg ggcttcctaa taccgcccat agccactccc ccattgacgt
     5581 caatggtctc tatatatggt ctttcctatt gacgtcatat gggcggtcct attgacgtat
     5641 atggcgcctc ccccattgac gtcaattacg gtaaatggcc cgcctggctc aatgcccatt
     5701 gacgtcaata ggaccaccca ccattgacgt caatgggatg gctcattgcc cattcatatc
     5761 cgttctcacg ccccctattg acgtcaatga cggtaaatgg cccacttggc agtacatcaa
     5821 tatctattaa tagtaacttg gcaagtacat tactattgga aggacgccag ggtacattgg
     5881 cagtactccc attgacgtca atggcggtaa atggcccgcg atggctgcca agtacatccc
     5941 cattgacgtc aatggggagg ggcaatgacg caaatgggcg ttccattgac gtaaatgggc
     6001 ggtaggcgtg cctaatggga ggtctatata agcaatgctc gtttag
//
