LOCUS       sequence-480246-        6610 bp    DNA     circular SYN 17-NOV-2025
DEFINITION  Drives expression of 3 different gRNAs targeting cmlc2 (myl7), and 
            expression of nuclear 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 Nov 17, 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_cmlc2(myl7)_cmlc2-nmKate
FEATURES             Location/Qualifiers
     source          1..6610
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     98..115
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    232..253
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(268..273,274..291,292..298)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    306..322
                     /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     311..333
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     330..346
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     330..346
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3025..3045
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3046..3744
                     /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      3746..3755
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4340..4357)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4340..4356)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4349..4371)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4565..4584)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4684..4706
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4744..4762)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4830..4934
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4935..5003,5004..5795)
                     /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(5153..5172)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5966..6554
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6455..6474
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
       61 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
      121 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
      181 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
      241 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
      301 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
      361 gcttgcatgg tccctttagc cagtagcggt tctaggcacg ggccgtccgg gcggtggcct
      421 ggggcggaaa actgaagggg ggcggcaccg gcggctcagc ccttgtaata tattatatgc
      481 accactattg gtttacttat gtcacagttt gtaagtttgt aacagcctga acctggatca
      541 gcttaggcct tcagtaagtc tctcagcgtt tctccagcct cggtcattca gtaagacgag
      601 tttcctgctc tcggcgtctt ttgttctggt cttcaaggag gggggaaatg ttctgcgcat
      661 gcctgtgggg gagggagaag gacacgtcac tgaaaacgtc cctgcatcac accgagacac
      721 ccaatcactc aagccaagac cagataattt tgcatatgct ttacagtttg aaaattacca
      781 cggtaatccc tcacacaaac tctggatttg agatccttca ggttttatca gtttgcaggt
      841 ttatgtcacc atgatatagg gtcagacttg atctaaggga gctgaataag tggtttagtc
      901 actcaccacc tcccaaaaac atacccagaa gtccctggta tatatagctc tccctccagc
      961 tcttggttcg ggctgcatag atcagaaccg tttaagagct atgctggaaa cagcatagca
     1021 agtttaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     1081 tagtcatctc attaccctcc acgtgtctgt ctgggttttc atgggctctg ctctagtgag
     1141 agcagcttcc ttttgtcgga gtgttttgtg ctcttatcca aggcaggggg gatctgcgca
     1201 tgcctgtggg gggaggagaa ggacacgtga acaaaagctc ctcgatgtca cacaggaagt
     1261 tcaggaactc atccaatcac tctaaagaaa cggcctgttt ccttcgcata cgcttacagc
     1321 tccaaaactc tacggtaaac ctacataaac tgctggtttt caaattttaa agaatttaag
     1381 ggtttacagg tttactacta cacagtgatt tactgacaca tgtaagtgta aatgagttga
     1441 ataagtaagt aagccatata ccacacatga aacacatacc cagaagtcac tggtatatat
     1501 agccgtcctc cagactccca gttcggttct ctccaaagag ggtggtttaa gagctatgct
     1561 ggaaacagca tagcaagttt aaataaggct agtccgttat caacttgaaa aagtggcacc
     1621 gagtcggtgc tttttttaaa ccaaatccaa tccgagagtc tgtgaatgtt tcgatgggcc
     1681 gtttgctggc gtctgagcat gttgtcctct ggtctgcggc gggggaaggt ccgcgcatgc
     1741 ctgtggggga aggcgaagga cacgaggaca aaagatcagt tacgtcacag aagaactcat
     1801 ccaatcacac aagcctaaaa ccagcttcct ctgcatatgc tttacagttc aaaaatacta
     1861 cggtaaacct ctacaaaact gctggtttca aactttgaag gttttaagcg tttgcaggtt
     1921 tgccccgaag aggtttactg tcatgtttga ggtaaatgag ttgaataagt aggtttatcc
     1981 acttaccaca tggcagaaac atacccagaa gtcccgggtt tatatagcag ttctccaggc
     2041 tcctagttcg gacaactcct gtggcattag tttaagagct atgctggaaa cagcatagca
     2101 agtttaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     2161 ttaaacctga ctccaaatca gcagacttaa cattctttaa aatgattgat tcaatagtga
     2221 taaaaatcag gcatagccag ttgtaacttt agataaatta cagaaaatgt caaatacaga
     2281 gaaccgattc ttttttatga tacatccaag cacacattta acacaatcca ggcaaacccc
     2341 gaatttcaca gtcacaagca ctgtttgtac aagagctttg cctaaggaca cacagtctct
     2401 ataagtccag gtcgttggtt tcactcttat tttaaacatg tgacattttt cctgccatcc
     2461 tgtcttaggc tgctgtttgc ttcattccat gtcacattaa tttcctcagt agcacctttt
     2521 acacacacag ccaatctttt ccagaaaatt caattgcttt gaaaagataa tgtgtgaaca
     2581 aatccattta gaaaaggaaa attaagaatt tgtaaaatca tctgtaaatt gttggcattc
     2641 ttctgtatat gaacatcaca tcatttacag gtaaaggtct ggtcattaat tatatgacaa
     2701 tttactggta ttattttgtg aaaggggcta ttttcaatgc attcatccat ccttttcatc
     2761 ccccaaatct ctcattcatg tccccctccc catctgcaca ctttatctca ttttccaccc
     2821 tgctggaatc tgagcacttg tgcagttatc agggctcctg tatttaggag gctctgggtg
     2881 tccatgtagg ggacgaacag aaacactgca gacctttata gaagaacaaa tgataagagt
     2941 cctcatacat aaagactcca ttagaaacgt cagtgaccca ggagcccaga ccaacagcaa
     3001 agcagacagt gaaccgccac catgccaaag aagaagcgta aggtagtgag cgagctgatt
     3061 aaggagaaca tgcacatgaa gctgtacatg gagggcaccg tgaacaacca ccacttcaag
     3121 tgcacatccg agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggcg
     3181 gtcgagggcg gccctctccc cttcgccttc gacatcctgg ctaccagctt catgtacggc
     3241 agcaaaacct tcatcaacca cacccagggc atccccgact tctttaagca gtccttcccc
     3301 gagggcttca catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc
     3361 caggacacca gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac
     3421 ttcccatcca acggccctgt gatgcagaag aaaacactcg gctgggaggc ctccaccgag
     3481 accctgtacc ccgctgacgg cggcctggaa ggcagagccg acatggccct gaagctcgtg
     3541 ggcgggggcc acctgatctg caacttgaag accacataca gatccaagaa acccgctaag
     3601 aacctcaaga tgcccggcgt ctactatgtg gacagaagac tggaaagaat caaggaggcc
     3661 gacaaagaga cctacgtcga gcagcacgag gtggctgtgg ccagatactg cgacctccct
     3721 agcaaactgg ggcacagagt ttaaagccac catggagatc tcgagactag taattaagtc
     3781 tcagccaccg ttaactgaac atgtcaaaac ctgtggagac tgttgagatt tgatgttctg
     3841 aaaagataaa gcctataaat aaaatgttgc ccaaatttcc tgcctgatgt ttttctttgt
     3901 ctttgctaca tggctttgct gctcggatcg gctcactctg tgtatgccac gttcactttg
     3961 tactctcctt ctcacggtag gtttattatt tttagatgtg cagttagttt ctgtgaaata
     4021 acacaccaca cactgatatt gtctgtgcat tgacttggtg agtgcacatt gtttttgatc
     4081 ttgacatatt tatatttgat tgatcaggtg aactgtgtga atctaaagtg ctccatacag
     4141 atgttctgca ttgaaaatat tctcatttta ttagtggaag tgagtgtatg ccagcggccg
     4201 cggtaccctg cagtctagac atttcagttg acgaagacaa acaaagttct gttgtgacta
     4261 tggggggggg gggcgcctgg ggatggtctc gcccggggag taattcaggg tagaaccgcc
     4321 actgccttta gcgacattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     4381 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     4441 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc
     4501 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc
     4561 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     4621 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4681 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     4741 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     4801 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4861 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4921 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4981 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5041 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5101 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5161 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5221 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5281 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5341 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5401 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5461 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5521 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     5581 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5641 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5701 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5761 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5821 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5881 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5941 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6001 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6061 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6121 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6181 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6241 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6301 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6361 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6421 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6481 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6541 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6601 cttttgctca
//
