LOCUS       sequence-472879-        6610 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Drives expression of 3 different gRNAs targeting amhc (myh6), and 
            expression of nclear mKate in cardiomyocytes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6610)
  AUTHORS   Keeley S, Fernandez-Lajarin M, Bergemann D, John N, Parrott L, 
            Andrea BE, Gonzalez-Rosa JM
  TITLE     Rapid and robust generation of cardiomyocyte-specific crispants in 
            zebrafish using the cardiodeleter system.
  JOURNAL   Cell Rep Methods. 2025 Mar 24;5(3):101003. doi: 
            10.1016/j.crmeth.2025.101003.
  PUBMED    40132543
REFERENCE   2  (bases 1 to 6610)
  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 Rep 
            Methods."; date: "2025-03-24"; volume: "5(3):101003. doi"; pages: " 
            10.1016/j.crmeth.2025.101003"
COMMENT     Sequence Label: pTol1-U6abc_amhc(myh6)_cmlc2-nmKate
FEATURES             Location/Qualifiers
     source          1..6610
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             20..40
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             41..739
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKA
                     VEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALK
                     LVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHRV"
     regulatory      741..750
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1335..1352)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1335..1351)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1344..1366)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1560..1579)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1679..1701
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1739..1757)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1825..1929
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1930..1998,1999..2790)
                     /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     complement(2148..2167)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2961..3549
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3450..3469
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3703..3720
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3837..3858
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3873..3878,3879..3896,3897..3903)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3911..3927
                     /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)."
     primer_bind     3916..3938
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3935..3951
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3935..3951
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 acagtgaacc gccaccatgc caaagaagaa gcgtaaggta gtgagcgagc tgattaagga
       61 gaacatgcac atgaagctgt acatggaggg caccgtgaac aaccaccact tcaagtgcac
      121 atccgagggc gaaggcaagc cctacgaggg cacccagacc atgagaatca aggcggtcga
      181 gggcggccct ctccccttcg ccttcgacat cctggctacc agcttcatgt acggcagcaa
      241 aaccttcatc aaccacaccc agggcatccc cgacttcttt aagcagtcct tccccgaggg
      301 cttcacatgg gagagagtca ccacatacga agacgggggc gtgctgaccg ctacccagga
      361 caccagcctc caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttccc
      421 atccaacggc cctgtgatgc agaagaaaac actcggctgg gaggcctcca ccgagaccct
      481 gtaccccgct gacggcggcc tggaaggcag agccgacatg gccctgaagc tcgtgggcgg
      541 gggccacctg atctgcaact tgaagaccac atacagatcc aagaaacccg ctaagaacct
      601 caagatgccc ggcgtctact atgtggacag aagactggaa agaatcaagg aggccgacaa
      661 agagacctac gtcgagcagc acgaggtggc tgtggccaga tactgcgacc tccctagcaa
      721 actggggcac agagtttaaa gccaccatgg agatctcgag actagtaatt aagtctcagc
      781 caccgttaac tgaacatgtc aaaacctgtg gagactgttg agatttgatg ttctgaaaag
      841 ataaagccta taaataaaat gttgcccaaa tttcctgcct gatgtttttc tttgtctttg
      901 ctacatggct ttgctgctcg gatcggctca ctctgtgtat gccacgttca ctttgtactc
      961 tccttctcac ggtaggttta ttatttttag atgtgcagtt agtttctgtg aaataacaca
     1021 ccacacactg atattgtctg tgcattgact tggtgagtgc acattgtttt tgatcttgac
     1081 atatttatat ttgattgatc aggtgaactg tgtgaatcta aagtgctcca tacagatgtt
     1141 ctgcattgaa aatattctca ttttattagt ggaagtgagt gtatgccagc ggccgcggta
     1201 ccctgcagtc tagacatttc agttgacgaa gacaaacaaa gttctgttgt gactatgggg
     1261 ggggggggcg cctggggatg gtctcgcccg gggagtaatt cagggtagaa ccgccactgc
     1321 ctttagcgac attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     1381 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     1441 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg
     1501 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt
     1561 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     1621 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     1681 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     1741 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     1801 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     1861 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     1921 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     1981 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     2041 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     2101 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     2161 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     2221 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     2281 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     2341 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     2401 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     2461 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     2521 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     2581 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     2641 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     2701 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     2761 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     2821 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     2881 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2941 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3001 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3061 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     3121 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3181 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3241 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3301 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3361 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3421 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     3481 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3541 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3601 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3661 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     3721 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     3781 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     3841 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     3901 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     3961 gccaagcttg catggtccct ttagccagta gcggttctag gcacgggccg tccgggcggt
     4021 ggcctggggc ggaaaactga aggggggcgg caccggcggc tcagcccttg taatatatta
     4081 tatgcaccac tattggttta cttatgtcac agtttgtaag tttgtaacag cctgaacctg
     4141 gatcagctta ggccttcagt aagtctctca gcgtttctcc agcctcggtc attcagtaag
     4201 acgagtttcc tgctctcggc gtcttttgtt ctggtcttca aggagggggg aaatgttctg
     4261 cgcatgcctg tgggggaggg agaaggacac gtcactgaaa acgtccctgc atcacaccga
     4321 gacacccaat cactcaagcc aagaccagat aattttgcat atgctttaca gtttgaaaat
     4381 taccacggta atccctcaca caaactctgg atttgagatc cttcaggttt tatcagtttg
     4441 caggtttatg tcaccatgat atagggtcag acttgatcta agggagctga ataagtggtt
     4501 tagtcactca ccacctccca aaaacatacc cagaagtccc tggtatatat agctctccct
     4561 ccagctcttg gttcgggcgc ctggaggagg cagggtttaa gagctatgct ggaaacagca
     4621 tagcaagttt aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     4681 ttttttagtc atctcattac cctccacgtg tctgtctggg ttttcatggg ctctgctcta
     4741 gtgagagcag cttccttttg tcggagtgtt ttgtgctctt atccaaggca ggggggatct
     4801 gcgcatgcct gtggggggag gagaaggaca cgtgaacaaa agctcctcga tgtcacacag
     4861 gaagttcagg aactcatcca atcactctaa agaaacggcc tgtttccttc gcatacgctt
     4921 acagctccaa aactctacgg taaacctaca taaactgctg gttttcaaat tttaaagaat
     4981 ttaagggttt acaggtttac tactacacag tgatttactg acacatgtaa gtgtaaatga
     5041 gttgaataag taagtaagcc atataccaca catgaaacac atacccagaa gtcactggta
     5101 tatatagccg tcctccagac tcccagttcg gggagcaggc cgagcccgag tttaagagct
     5161 atgctggaaa cagcatagca agtttaaata aggctagtcc gttatcaact tgaaaaagtg
     5221 gcaccgagtc ggtgcttttt ttaaaccaaa tccaatccga gagtctgtga atgtttcgat
     5281 gggccgtttg ctggcgtctg agcatgttgt cctctggtct gcggcggggg aaggtccgcg
     5341 catgcctgtg ggggaaggcg aaggacacga ggacaaaaga tcagttacgt cacagaagaa
     5401 ctcatccaat cacacaagcc taaaaccagc ttcctctgca tatgctttac agttcaaaaa
     5461 tactacggta aacctctaca aaactgctgg tttcaaactt tgaaggtttt aagcgtttgc
     5521 aggtttgccc cgaagaggtt tactgtcatg tttgaggtaa atgagttgaa taagtaggtt
     5581 tatccactta ccacatggca gaaacatacc cagaagtccc gggtttatat agcagttctc
     5641 caggctccta gttcgggcag gagttgcatc cgttgtttaa gagctatgct ggaaacagca
     5701 tagcaagttt aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     5761 tttttttaaa cctgactcca aatcagcaga cttaacattc tttaaaatga ttgattcaat
     5821 agtgataaaa atcaggcata gccagttgta actttagata aattacagaa aatgtcaaat
     5881 acagagaacc gattcttttt tatgatacat ccaagcacac atttaacaca atccaggcaa
     5941 accccgaatt tcacagtcac aagcactgtt tgtacaagag ctttgcctaa ggacacacag
     6001 tctctataag tccaggtcgt tggtttcact cttattttaa acatgtgaca tttttcctgc
     6061 catcctgtct taggctgctg tttgcttcat tccatgtcac attaatttcc tcagtagcac
     6121 cttttacaca cacagccaat cttttccaga aaattcaatt gctttgaaaa gataatgtgt
     6181 gaacaaatcc atttagaaaa ggaaaattaa gaatttgtaa aatcatctgt aaattgttgg
     6241 cattcttctg tatatgaaca tcacatcatt tacaggtaaa ggtctggtca ttaattatat
     6301 gacaatttac tggtattatt ttgtgaaagg ggctattttc aatgcattca tccatccttt
     6361 tcatccccca aatctctcat tcatgtcccc ctccccatct gcacacttta tctcattttc
     6421 caccctgctg gaatctgagc acttgtgcag ttatcagggc tcctgtattt aggaggctct
     6481 gggtgtccat gtaggggacg aacagaaaca ctgcagacct ttatagaaga acaaatgata
     6541 agagtcctca tacataaaga ctccattaga aacgtcagtg acccaggagc ccagaccaac
     6601 agcaaagcag
//
