LOCUS       sequence-480244-        6610 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Drives expression of 3 different gRNAs targeting ect2, 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 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_ect2_cmlc2-nmKate
FEATURES             Location/Qualifiers
     source          1..6610
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(142..147,148..165,166..172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /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     185..207
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     204..220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2899..2919
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2920..3618
                     /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      3620..3629
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4214..4231)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4214..4230)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4223..4245)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4439..4458)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4558..4580
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4618..4636)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4704..4808
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4809..4877,4878..5669)
                     /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(5027..5046)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5840..6428
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6329..6348
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6582..6599
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc
      241 atggtccctt tagccagtag cggttctagg cacgggccgt ccgggcggtg gcctggggcg
      301 gaaaactgaa ggggggcggc accggcggct cagcccttgt aatatattat atgcaccact
      361 attggtttac ttatgtcaca gtttgtaagt ttgtaacagc ctgaacctgg atcagcttag
      421 gccttcagta agtctctcag cgtttctcca gcctcggtca ttcagtaaga cgagtttcct
      481 gctctcggcg tcttttgttc tggtcttcaa ggagggggga aatgttctgc gcatgcctgt
      541 gggggaggga gaaggacacg tcactgaaaa cgtccctgca tcacaccgag acacccaatc
      601 actcaagcca agaccagata attttgcata tgctttacag tttgaaaatt accacggtaa
      661 tccctcacac aaactctgga tttgagatcc ttcaggtttt atcagtttgc aggtttatgt
      721 caccatgata tagggtcaga cttgatctaa gggagctgaa taagtggttt agtcactcac
      781 cacctcccaa aaacataccc agaagtccct ggtatatata gctctccctc cagctcttgg
      841 ttcgggcagg aggccccagg atagtttaag agctatgctg gaaacagcat agcaagttta
      901 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttagtca
      961 tctcattacc ctccacgtgt ctgtctgggt tttcatgggc tctgctctag tgagagcagc
     1021 ttccttttgt cggagtgttt tgtgctctta tccaaggcag gggggatctg cgcatgcctg
     1081 tggggggagg agaaggacac gtgaacaaaa gctcctcgat gtcacacagg aagttcagga
     1141 actcatccaa tcactctaaa gaaacggcct gtttccttcg catacgctta cagctccaaa
     1201 actctacggt aaacctacat aaactgctgg ttttcaaatt ttaaagaatt taagggttta
     1261 caggtttact actacacagt gatttactga cacatgtaag tgtaaatgag ttgaataagt
     1321 aagtaagcca tataccacac atgaaacaca tacccagaag tcactggtat atatagccgt
     1381 cctccagact cccagttcgg aggtgggcgg ccccattcgt ttaagagcta tgctggaaac
     1441 agcatagcaa gtttaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     1501 gtgctttttt taaaccaaat ccaatccgag agtctgtgaa tgtttcgatg ggccgtttgc
     1561 tggcgtctga gcatgttgtc ctctggtctg cggcggggga aggtccgcgc atgcctgtgg
     1621 gggaaggcga aggacacgag gacaaaagat cagttacgtc acagaagaac tcatccaatc
     1681 acacaagcct aaaaccagct tcctctgcat atgctttaca gttcaaaaat actacggtaa
     1741 acctctacaa aactgctggt ttcaaacttt gaaggtttta agcgtttgca ggtttgcccc
     1801 gaagaggttt actgtcatgt ttgaggtaaa tgagttgaat aagtaggttt atccacttac
     1861 cacatggcag aaacataccc agaagtcccg ggtttatata gcagttctcc aggctcctag
     1921 ttcgggctca cttggaccga gtcgtttaag agctatgctg gaaacagcat agcaagttta
     1981 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttaaac
     2041 ctgactccaa atcagcagac ttaacattct ttaaaatgat tgattcaata gtgataaaaa
     2101 tcaggcatag ccagttgtaa ctttagataa attacagaaa atgtcaaata cagagaaccg
     2161 attctttttt atgatacatc caagcacaca tttaacacaa tccaggcaaa ccccgaattt
     2221 cacagtcaca agcactgttt gtacaagagc tttgcctaag gacacacagt ctctataagt
     2281 ccaggtcgtt ggtttcactc ttattttaaa catgtgacat ttttcctgcc atcctgtctt
     2341 aggctgctgt ttgcttcatt ccatgtcaca ttaatttcct cagtagcacc ttttacacac
     2401 acagccaatc ttttccagaa aattcaattg ctttgaaaag ataatgtgtg aacaaatcca
     2461 tttagaaaag gaaaattaag aatttgtaaa atcatctgta aattgttggc attcttctgt
     2521 atatgaacat cacatcattt acaggtaaag gtctggtcat taattatatg acaatttact
     2581 ggtattattt tgtgaaaggg gctattttca atgcattcat ccatcctttt catcccccaa
     2641 atctctcatt catgtccccc tccccatctg cacactttat ctcattttcc accctgctgg
     2701 aatctgagca cttgtgcagt tatcagggct cctgtattta ggaggctctg ggtgtccatg
     2761 taggggacga acagaaacac tgcagacctt tatagaagaa caaatgataa gagtcctcat
     2821 acataaagac tccattagaa acgtcagtga cccaggagcc cagaccaaca gcaaagcaga
     2881 cagtgaaccg ccaccatgcc aaagaagaag cgtaaggtag tgagcgagct gattaaggag
     2941 aacatgcaca tgaagctgta catggagggc accgtgaaca accaccactt caagtgcaca
     3001 tccgagggcg aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggcggtcgag
     3061 ggcggccctc tccccttcgc cttcgacatc ctggctacca gcttcatgta cggcagcaaa
     3121 accttcatca accacaccca gggcatcccc gacttcttta agcagtcctt ccccgagggc
     3181 ttcacatggg agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac
     3241 accagcctcc aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttccca
     3301 tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg aggcctccac cgagaccctg
     3361 taccccgctg acggcggcct ggaaggcaga gccgacatgg ccctgaagct cgtgggcggg
     3421 ggccacctga tctgcaactt gaagaccaca tacagatcca agaaacccgc taagaacctc
     3481 aagatgcccg gcgtctacta tgtggacaga agactggaaa gaatcaagga ggccgacaaa
     3541 gagacctacg tcgagcagca cgaggtggct gtggccagat actgcgacct ccctagcaaa
     3601 ctggggcaca gagtttaaag ccaccatgga gatctcgaga ctagtaatta agtctcagcc
     3661 accgttaact gaacatgtca aaacctgtgg agactgttga gatttgatgt tctgaaaaga
     3721 taaagcctat aaataaaatg ttgcccaaat ttcctgcctg atgtttttct ttgtctttgc
     3781 tacatggctt tgctgctcgg atcggctcac tctgtgtatg ccacgttcac tttgtactct
     3841 ccttctcacg gtaggtttat tatttttaga tgtgcagtta gtttctgtga aataacacac
     3901 cacacactga tattgtctgt gcattgactt ggtgagtgca cattgttttt gatcttgaca
     3961 tatttatatt tgattgatca ggtgaactgt gtgaatctaa agtgctccat acagatgttc
     4021 tgcattgaaa atattctcat tttattagtg gaagtgagtg tatgccagcg gccgcggtac
     4081 cctgcagtct agacatttca gttgacgaag acaaacaaag ttctgttgtg actatggggg
     4141 gggggggcgc ctggggatgg tctcgcccgg ggagtaattc agggtagaac cgccactgcc
     4201 tttagcgaca ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     4261 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     4321 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc
     4381 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta
     4441 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     4501 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     4561 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     4621 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     4681 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     4741 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     4801 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     4861 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     4921 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     4981 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     5041 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     5101 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     5161 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     5221 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     5281 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     5341 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     5401 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     5461 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     5521 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     5581 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     5641 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     5701 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     5761 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     5821 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     5881 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     5941 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     6001 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6061 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6121 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6181 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     6241 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     6301 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     6361 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     6421 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     6481 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     6541 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     6601 aagcggaaga
//
