LOCUS       sequence-461280-        4538 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bigMamAct is evolution of biGBac for mammalian cells. Has empty 
            EF1alpha-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 4538)
  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 4538)
  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 EF1alpha
FEATURES             Location/Qualifiers
     source          1..4538
                     /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        2473..3651
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2704..3642
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3599..3619
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     polyA_signal    3938..4072
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3975..3994)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4029..4048
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  4182..4347
                     /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 atagatcgaa ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc
     2521 gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta
     2581 aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg
     2641 tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca
     2701 caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc
     2761 gtgccttgaa ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt
     2821 ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga
     2881 gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc
     2941 tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg
     3001 ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg
     3061 gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg
     3121 cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct
     3181 gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc
     3241 cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc
     3301 tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa
     3361 agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc
     3421 aggcacctcg attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg
     3481 ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg
     3541 cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc
     3601 aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg aggaattctg
     3661 cagtcgatcg acggtaccgc gggcccgccc gggataccgt cccaccatcg ggcgcggatc
     3721 ccggtccgaa gcgcgcggaa ttcaaaggcc tacgtcgacg agctcactag tcgcggccgc
     3781 tttcgaatct agagcctgca gtctcgacaa gcttgtcgag aagtactaga ggatcataat
     3841 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     3901 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     3961 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4021 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tctgatcact
     4081 gcttgagcct agaagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     4141 ccaccgctga gcaataacta tcataacccc taggagatcc gaaccagata agtgaaatct
     4201 agttccaaac tattttgtca tttttaattt tcgtattagc ttacgacgct acacccagtt
     4261 cccatctatt ttgtcactct tccctaaata atccttaaaa actccatttc cacccctccc
     4321 agttcccaac tattttgtcc gcccacagcg gggcattttt cttcctgtta tgtttttaat
     4381 caaacatcct gccaactcca tgtgacaaac cgtcatcttc ggctactttt tctctgtcac
     4441 agaatgaaaa tttttctgtc atctcttcgt tattaatgtt tgtaattgac tgaatatcaa
     4501 cgcttatttg cagcctgaat ggcgaatggg acgcgccc
//
