LOCUS       sequence-326578-        2526 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA synthesis vector for ttn.1_C3 (zebrafish titin .1)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2526)
  AUTHORS   Hall TE, Martel N, Ariotti N, Xiong Z, Lo HP, Ferguson C, Rae J, Lim
            YW, Parton RG
  TITLE     In vivo cell biological screening identifies an endocytic capture 
            mechanism for T-tubule formation.
  JOURNAL   Nat Commun. 2020 Jul 24;11(1):3711. doi: 10.1038/s41467-020-17486-w.
  PUBMED    32709891
REFERENCE   2  (bases 1 to 2526)
  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: "Nat 
            Commun."; date: "2020-07-24"; volume: "11(1):3711. doi"; pages: " 
            10.1038/s41467-020-17486-w"
COMMENT     Sequence Label: pT7gRNA-ttn.1_C3
FEATURES             Location/Qualifiers
     source          1..2526
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      173..761
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     662..681
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     915..932
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1049..1070
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1085..1090,1091..1108,1109..1115)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1123..1139
                     /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     1128..1150
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1147..1163
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1147..1163
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1187..1205
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        1224..1299
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1327..1344)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1327..1343)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1336..1358)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        1563..1667
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1668..1736,1737..2526,1..2)
                     /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(1886..1905)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
       61 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
      121 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
      181 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
      241 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
      301 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
      361 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
      421 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
      481 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
      541 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
      601 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
      661 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
      721 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
      781 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
      841 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
      901 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
      961 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     1021 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     1081 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     1141 ttcacacagg aaacagctat gaccatgatt acgccaagct tgaaattaat acgactcact
     1201 ataggtactg gggaattcag tgagttttag agctagaaat agcaagttaa aataaggcta
     1261 gtccgttatc aacttgaaaa agtggcaccg agtcggtgcg gatccccggg taccgagctc
     1321 gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     1381 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     1441 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt
     1501 tctccttacg catctgtgcg gtatttcaca ccgcataagg tggcactttt cggggaaatg
     1561 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     1621 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     1681 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     1741 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     1801 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     1861 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     1921 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     1981 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     2041 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     2101 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     2161 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     2221 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     2281 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     2341 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     2401 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     2461 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     2521 attggt
//
