LOCUS       sequence-461573-        3549 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bigMamAct is evolution of biGBac for mammalian cells. Has empty 
            U6-driven cassette; users should clone gene of interest into shuttle
            plasmid prior to multi-assembly step by Gibson cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3549)
  AUTHORS   Pelosse M, Marcia M
  TITLE     biGMamAct: efficient CRISPR/Cas9-mediated docking of large 
            functional DNA cargoes at the ACTB locus
  JOURNAL   Synthetic Biology 2025.02.13
REFERENCE   2  (bases 1 to 3549)
  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: "Synthetic 
            Biology 2025.02.13"
COMMENT     Sequence Label: psLIB U6
FEATURES             Location/Qualifiers
     source          1..3549
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      54..642
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     543..562
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     796..813
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  947..1171
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(1238..1771)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(1960..1965,1966..1982,1983..1988))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    complement(2384..2417)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2468..2716
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2468..2488
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2639..2658
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2735..2810
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     polyA_signal    2949..3083
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2986..3005)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3040..3059
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  3193..3358
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
ORIGIN
        1 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
       61 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
      121 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
      181 gcgcagatac caaatactat tcttctagtg tagccgtagt taggccacca cttcaagaac
      241 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
      301 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
      361 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
      421 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
      481 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
      541 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
      601 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
      661 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
      721 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
      781 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
      841 tttctcctta cgcatctgtg cggtatttca caccgcatag accagccgcg taacctggca
      901 aaatcggtta cggttgagta ataaatggat gccctgcgta agcgggtgtg ggcggacaat
      961 aaagtcttaa actgaacaaa atagatctaa actatgacaa taaagtctta aactagacag
     1021 aatagttgta aactgaaatc agtccagtta tgctgtgaaa aagcatactg gacttttgtt
     1081 atggctaaag caaactcttc attttctgaa gtgcaaattg cccgtcgtat taaagagggg
     1141 cgtggccaag ggcatggtaa agactatatt cgcggcgttg tgacaattta ccgaacaact
     1201 ccgcggccgg gaagccgatc tcggcttgaa cgaattgtta ggtggcggta cttgggtcga
     1261 tatcaaagtg catcacttct tcccgtatgc ccaactttgt atagagagcc actgcgggat
     1321 cgtcaccgta atctgcttgc acgtagatca cataagcacc aagcgcgttg gcctcatgct
     1381 tgaggagatt gatgagcgcg gtggcaatgc cctgcctccg gtgctcgccg gagactgcga
     1441 gatcatagat atagatctca ctacgcggct gctcaaactt gggcagaacg taagccgcga
     1501 gagcgccaac aaccgcttct tggtcgaagg cagcaagcgc gatgaatgtc ttactacgga
     1561 gcaagttccc gaggtaatcg gagtccggct gatgttggga gtaggtggct acgtctccga
     1621 actcacgacc gaaaagatca agagcagccc gcatggattt gacttggtca gggccgagcc
     1681 tacatgtgcg aatgatgccc atacttgagc cacctaactt tgttttaggg cgactgccct
     1741 gctgcgtaac atcgttgctg ctgcgtaaca tcgttgctgc tccataacat caaacatcga
     1801 cccacggcgt aacgcgcttg ctgcttggat gcccgaggca tagactgtac aaaaaaacag
     1861 tcataacaag ccatgaaaac cgccactgcg ccgttaccac cgctgcgttc ggtcaaggtt
     1921 ctggaccagt tgcgtgagcg catacgctac ttgcattaca gtttacgaac cgaacaggct
     1981 tatgtcaact gggtttgtgc cttcatccgt ttccacggtg tgcgtcaccc ggcaaccttg
     2041 ggcagcagcg aagtcgaggc atttctgtcc tggctggcga acgagcgcaa ggtttcggtc
     2101 tccacgcatc gtcaggcatt ggcggccttg ctgttcttct acggcaaggt gctgtgcacg
     2161 gatctgccct tgcttcagga gatcggtaga cctcggccgt cgcggcgctt gccggtggtg
     2221 ctgaccccgg atgaagtggt tcgcatcctc ggttttctgg aaggcgagca tcgtttgttc
     2281 gcccaggact ctagctatag ttctagtggt tggctacagc tttgtttgta ctatcaacag
     2341 gttgaactgc tgatcaacag atcctctacg cggccgcggt accataactt cgtatagcat
     2401 acattatacg aagttatctg gttttactag tatcgattcg cgacctactc cggaatatta
     2461 atagatcgag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     2521 tagagagata attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     2581 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     2641 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
     2701 gaaaggacga aacaccgggt cttcgagaag acctgtttta gagctagaaa tagcaagtta
     2761 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttgttt
     2821 tagagctaga aatagcaagt taaaataagg ctagtccgtt tttagcgcgt gcgccaattc
     2881 tgcagacaaa tggctctaga cacctccccc tgaacctgaa acataaaatg aatgcaattg
     2941 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3001 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3061 atgtatctta tcatgtctgg atctgatcac tgcttgagcc tagaagatcc ggctgctaac
     3121 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact atcataaccc
     3181 ctaggagatc cgaaccagat aagtgaaatc tagttccaaa ctattttgtc atttttaatt
     3241 ttcgtattag cttacgacgc tacacccagt tcccatctat tttgtcactc ttccctaaat
     3301 aatccttaaa aactccattt ccacccctcc cagttcccaa ctattttgtc cgcccacagc
     3361 ggggcatttt tcttcctgtt atgtttttaa tcaaacatcc tgccaactcc atgtgacaaa
     3421 ccgtcatctt cggctacttt ttctctgtca cagaatgaaa atttttctgt catctcttcg
     3481 ttattaatgt ttgtaattga ctgaatatca acgcttattt gcagcctgaa tggcgaatgg
     3541 gacgcgccc
//
