LOCUS       sequence-234781-        8563 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Zebrafish ubiquitin promoter driving Com-KRAB with Cerulean 
            reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8563)
  AUTHORS   Chong-Morrison V, Mayes S, Simoes FC, Senanayake U, Carroll DS, 
            Riley PR, Wilson SW, Sauka-Spengler T
  TITLE     Ac/Ds transposition for CRISPR/dCas9-SID4x epigenome modulation in 
            zebrafish.
  JOURNAL   Biol Open. 2023 Jun 15;12(6):bio059995. doi: 10.1242/bio.059995. 
            Epub 2023 Jun 27.
  PUBMED    37367831
REFERENCE   2  (bases 1 to 8563)
  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: "Biol 
            Open."; date: "2023-06-15"; volume: "12(6):bio059995. doi"; pages: "
            10.1242/bio.059995. Epub 2023 Jun 27"
COMMENT     Sequence Label: pVC-Ds-ubb:Com-KRAB-2a-Cerulean-Ds
FEATURES             Location/Qualifiers
     source          1..8563
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..22
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6..22
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     32..51
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        32..50
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      3828..3837
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3838..3858
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4123..4308
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             4324..4377
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(4390..4392,4393..5106)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4432..4453)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4693..4712)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5040..5061
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5158..5279
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5169..5188)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5223..5242
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5781..5801)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5781..5799)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5820..5836)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5820..5836)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5833..5855)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5844..5860
                     /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(5868..5874,5875..5892,5893..5898))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5913..5934
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6051..6068)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6222..6810)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6302..6321)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6981..7772,7773..7841))
                     /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     7604..7623
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7842..7946)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7972..8427)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8109..8130)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8321..8340
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(8554..8563,1..13)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattggg
       61 tacccgacgc agggatgaaa gtaggatggg aaaatcccgt accgaccgtt atcgtataac
      121 cgattttgtt agttttatcc cgatcgattt cgaacccgag gtaaaaaacg aaaacggaac
      181 ggaaacggga tatacaaaac ggtaaacgga aacggaaacg gtagagctag tttcccgacc
      241 gtttcaccgg gatcccgttt ttaatcggga tgatcccgtt tcgttaccgt attttctaat
      301 tcgggatgac tgcaacagat ccctcgactc gagaccagca aagttctaga atttgtcgaa
      361 acatttatgt tatatatttc ctgaaaaaaa ttctgagtaa gttcttaagt gttattgcca
      421 gcaacataaa caacagacgg caaaatgaat aaatgataac aaagcagtag gcttaaataa
      481 acctaatttt tataggctgt tctctacaac cctcaaacag tgattagttt tgtacttata
      541 aacttgccct ttcattcata tttcaagaaa attggttcag aagatctgga tattctagca
      601 gttgttcaag ctcatggagg gatcagtgac ctgattccac aatgactagg cctaatccag
      661 aaattagatg actgtcaaca taaaaaggca cagcactcac tagctgccct atatatttta
      721 ttatatttta catatattat tttatttatt tagctctgag tgctgtactt tctggttaaa
      781 gaaaactgct tacaacagct aacctgtact acctcaggct cagggaattt ggaacaggtt
      841 tgtctggttt gtttctttaa ccatgcatgc ttgttttcaa ctatggcaac acagtcacat
      901 gggacattac agaaatgatt tgtcgatgac atgcgacttt tctttaataa agcgcaaaga
      961 tcccaaaaag caaactttta acaaaaatca tataattata ttttcaatcc agctttgtag
     1021 caactttgtg ctgctgttca ctcagcaaca gatagtcagt ataaggtcag tgtgtctcaa
     1081 agcagtgcca tctgtttcac acattgcgtt ctatatataa gtgtgctggt tgacacgaca
     1141 ctgtataagg cctaggctaa aacacaaaca atgtagaatg acactgtgtt ttttttgtaa
     1201 acaaatgttg tttttggtta aacatctttg tgaaaacatc ctcctgtcat gtatttgcta
     1261 tattcaaatg ttaaacccgt gcagaataga acatatacaa aaaaaaacaa cacaacacat
     1321 ttttaaacat tattaaatat caagtattgc tggcagttct gtttctgttt tacagtaccc
     1381 tttgccacag ttctccgctt ttcctggtcc agattccaca agtctgattc accaatagca
     1441 aagcgaataa acaaccaaag cagccaatca ctgcttgtag actgtcctgc gagaccggcc
     1501 cattccagca cattctggaa acttccttta tatgataatt ataaatacat ttaaattatt
     1561 gatacaaaac atgtaattcc tagaacataa ccatagcaat cattagtttt cagggtaatt
     1621 atgtattttt aggatttgac tgcggaaaga tctggtcatg tgacgtctca tgaacgtcac
     1681 ggccctgggt ttctataaat acagtaggac tctcgaccat cggcagattt ttcgaagaag
     1741 aagatcagtt tcaggagccg tactgttccg tttcaacgca aatattaacg gtaagagcga
     1801 atttcctagt ttgttttcat gccattcttt aaaaccatag cgtattactt taattatagt
     1861 aaactttcgc tttctttatt acaagagacg ttttgtgttg attctccgcg gacattttcg
     1921 gtcagacaat cagaaaatga ccgcggagga ccagtaactt gcattacacg taagttaaat
     1981 cttcgtgtat taaaatggtt aggttgttaa cgtcaaatag gttaccgtgt ttgcgtgtga
     2041 tcaggttggt tttgttagat ttttgtcagt atttttaatt tatttgtttt agtttattta
     2101 ttttttttgc tgaatcatag tttgtgaaca aagaacccgg atgttacata cagtacagcc
     2161 gccatgttac agagagttat aacttaatca ttttaaaaat aattttgcct tacttttagt
     2221 ttgtcatgtt gagaaatgag gaaatgttaa aatgaggaaa tatccaatta atttaatata
     2281 tcaaaataat ccatgattac aatgcactga actggagaaa attaagatgt tttctagtgt
     2341 catgaaacaa atgtaagaga tgtacattgt agatgtttta tgtcaagaat tggctagttg
     2401 atgcagcata ctggcgatac tcagttgtaa taacagtaac gttacatgtt aatagactac
     2461 tgagtatgct gttctgtcta tgtatgctct gtaagctacg agaaggactt ttttaaacag
     2521 taaagggtgc aatattttta caaattgaat taaataaagg ctgtctatta agtaatatgc
     2581 ttgatatttt tcttacttga tcggaaataa gaaaaaatat aaacgttgtt gctctaaaaa
     2641 tcctagttca gtttagccaa ccacaaatac ctttttgttc ctccaacagt ttttttttct
     2701 tctctataat atttggcagt ctatagtact ccaaatgttt ccccacagtc taactaattg
     2761 gtacagccaa aatcatgaca cttattgcaa taataatttt ggttcattgg cattgttgat
     2821 agcctgtgcc actaatatgg tcgattgatc atgcttcagg aagaaaacta tattgtttga
     2881 tgtaagatta ttaaatcttc acctgcttcc attacaaact attcccatct tattgaattc
     2941 tggtatgtct taaaggatta gttcacttcc caaatcaaaa tttacttagc cttttttcat
     3001 ccatgatccc tttttttcat cattaatgaa gaaattgttt ttgaaaaagt ttcaagattt
     3061 ttttctctat attgtggagc ttgttagttt aaaattccaa aatgcaatat gtggcttcaa
     3121 atggttctaa atgatcccag tcaaggaata acagtcttat ctaatgaaac cattagacct
     3181 ttaaaaataa aaaaaataaa agtatttatt tttaaatgac tgagtgatta agttgaattt
     3241 cagcgtttcc ttactgtgta gaagtccttc cttactggcc ccaccctttg gttctctgcc
     3301 aatctgctac ctaatgtaat gttgtggaac attattattc tttatttctt aattttttat
     3361 tttttatttt aaaacaatgt aaactgcaca gatgtgcagt ttgtttaaaa tggccaatgc
     3421 tttggaaatg catgacataa ttagatttca tgatgcacaa agccaaatct cagagcttgt
     3481 gcaaaatgag ctatcatttc actaggtaag accctaaatt ttcatatagg atcatttgga
     3541 caattttgct gcaggtaaaa tgcattctat agtccactgt cagccattgt tttggatagt
     3601 atttattttt ctctacaagt atagtcaata gttttctatt attttaaagg tttgtaacat
     3661 ttaagggtga ccaaatgcaa agtaaaattt cattttcggg tgaactatct cgtttaacat
     3721 gggagaagtg caaaacatac attattggct agaacattgt agtatttttt aaatggaaat
     3781 gtgtgattgc taatcttact ttgaatttgt ttacaggtac cgctagcgcc accatggccc
     3841 aaaaagaaaa gaaaagtggg tagtatgaaa tcaattcgct gtaaaaactg caacaaactg
     3901 ttatttaagg cggatagttt tgatcacatt gaaatcaggt gtccgcgttg caaacgtcac
     3961 atcataatgc tgaatgcctg cgagcatccc acggagaaac attgtgggaa aagagaaaaa
     4021 atcacgcatt ctgacgaaac cgtgcgttat ggctccacta gtgggcacaa gcttaatggc
     4081 ggtggcggag ggatggatgc taagtcacta actgcctggt cccggacact ggtgaccttc
     4141 aaggatgtat ttgtggactt caccagggag gagtggaagc tgctggacac tgctcagcag
     4201 atcgtgtaca gaaatgtgat gctggagaac tataagaacc tggtttcctt gggttatcag
     4261 cttactaagc cagatgtgat cctccggttg gagaagggag aagagcccga tatcggatca
     4321 ggagagggca ggggaagtct tctaacatgc ggggacgtgg aggaaaatcc cgggccccgg
     4381 tcgcgatcgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     4441 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     4501 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     4561 cccaccctcg tgaccaccct gacctggggc gtgcagtgct tcgcccgcta ccccgaccac
     4621 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     4681 atcttcttca aagacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     4741 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     4801 gggcacaagc tggagtacaa cgccatcagc gacaacgtct atatcaccgc cgacaagcag
     4861 aagaacggca tcaaggccaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     4921 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     4981 aaccactacc tgagcaccca gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac
     5041 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     5101 aagtaaagcg gccgcgactc tagagtcggg gcggccggcc gcttcgagca gacatgataa
     5161 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     5221 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     5281 acaacaacaa ttgcattcat tttatgtttc gggttcaggg ggaggtgtgg gaggtttttt
     5341 aaagcaagta aaacctctac aaatgtggta ccatggtcta gaagtagact tatatggctt
     5401 cttatgttag ccaagagccc aagacttatc acttatgtgc tacattaaac tatgtgtgct
     5461 ccagatttat atggatttta tctatgttta attaagactt gtgtttacaa ttttttatat
     5521 ttgtttttaa gttttgaata tatgttttca tgtgtgattt taccgaacaa aaataccggt
     5581 tcccgtccga tttcgacttt aacccgaccg gatcgtatcg gttttcgatt accgtattta
     5641 tcccgttcgt tttcgttacc ggtatatccc gttttcgttt ccgtcccgca agttaaatat
     5701 gaaaatgaaa acggtagagg tattttaccg accgttaccg accgttttca tccctagtcg
     5761 gggagctcca gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg
     5821 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5881 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5941 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     6001 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     6061 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     6121 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     6181 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     6241 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     6301 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     6361 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     6421 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     6481 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     6541 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     6601 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6661 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6721 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6781 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6841 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6901 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6961 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     7021 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     7081 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     7141 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     7201 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     7261 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     7321 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     7381 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     7441 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     7501 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     7561 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     7621 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     7681 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     7741 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     7801 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     7861 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     7921 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     7981 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     8041 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     8101 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     8161 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     8221 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     8281 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     8341 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     8401 cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg
     8461 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
     8521 caaggcgatt aagttgggta acgccagggt tttcccagtc acg
//
