LOCUS       sequence-461256-        5463 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Harbor sgRNA+PAM sequence of ActinB gene for knockin using CRISPR 
            and Homology Independent Targeted Integration. Contains last ActB 
            exon fused T2A-tet regulatory elemeny-P2A-puro and Hygro.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5463)
  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 5463)
  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: psBIG1a ActBT2ATetP2AP - H
FEATURES             Location/Qualifiers
     source          1..5463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             241..294
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(295..912,913..918,919..954,955..957,958..993,994..996,
                     997..1032,1033..1038)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             1039..1095
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1099..1698
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1099..1118)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     polyA_signal    1747..1881
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     1838..1857
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1952..1985
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        join(2013..2018,2019..2035,2036..2041)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             2211..2744
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     rep_origin      complement(2804..3392)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2884..2903)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3735..3816
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(3864..4889)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(3864..4889)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(4963..5463)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5026..5044)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
ORIGIN
        1 atgagacagc tccccacaca ccacagggtg tgtggggagc tgtcacatcc agggtcctca
       61 ctgcctgtcc ccttccctcc tcagatcatt gctcctcctg agcgcaagta ctccgtgtgg
      121 atcggcggct ccatcctggc ctcgctgtcc accttccagc agatgtggat cagcaagcag
      181 gagtatgacg agtccggccc ctccatcgtc caccgcaaat gcttcggggc cggatcggga
      241 gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg ccccatgtct
      301 agactggaca agagcaaagt cataaacggc gctctggaat tactcaatgg agtcggtatc
      361 gaaggcctga cgacaaggaa actcgctcaa aagctgggag ttgagcagcc taccctgtac
      421 tggcacgtga agaacaagcg ggccctgctc gatgccctgc caatcgagat gctggacagg
      481 catcataccc acttctgccc cctggaaggc gagtcatggc aagactttct gcggaacaac
      541 gccaagtcat tccgctgtgc tctcctctca catcgcgacg gggctaaagt gcatctcggc
      601 acccgcccaa cagagaaaca gtacgaaacc ctggaaaatc agctcgcgtt cctgtgtcag
      661 caaggcttct ccctggagaa cgcactgtac gctctgtccg ccgtgggcca ctttacactg
      721 ggctgcgtat tggaggaaca ggagcatcaa gtagcaaaag aggaaagaga gacacctacc
      781 accgattcta tgcccccact tctgagacaa gcaattgagc tgttcgaccg gcagggagcc
      841 gaacctgcct tccttttcgg cctggaacta atcatatgtg gcctggagaa acagctaaag
      901 tgcgaaagcg gcgggccggc cgacgccctt gacgattttg acttagacat gctcccagcc
      961 gatgcccttg acgactttga ccttgatatg ctgcctgctg acgctcttga cgattttgac
     1021 cttgacatgc tccccggggc cacgaacttc tctctgttaa agcaagcagg agacgtggaa
     1081 gaaaaccccg gtcctgggat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     1141 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     1201 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     1261 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     1321 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     1381 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     1441 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     1501 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     1561 ggggtgcccg ccttcctgga gacctccgcg ccccacaacc tccccttcta cgagcggctc
     1621 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     1681 cgcaagcccg gtgcctgaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     1741 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     1801 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     1861 gtatcttatc atgtctggat ctgatcactg cttgttccgt gtttcagtta gcctcgataa
     1921 aaactttaga ccatagagcg tttaaaccca gataacttcg tataatgtat gctatacgaa
     1981 gttatggtac cgcggcgaag gcacgaaccc agttgacata agcctgttcg gttcgtaaac
     2041 tgtaatgcaa gtagcgtatg cgctcacgca actggtccag aaccttgacc gaacgcagcg
     2101 gtggtaacgg cgcagtggcg gttttcatgg cttgttatga ctgttttttt gtacagtcta
     2161 tgcctcgggc atccaagcag caagcgcgtt acgccgtggg tcgatgtttg atgttatgga
     2221 gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaggt ggctcaagta
     2281 tgggcatcat tcgcacatgt aggctcggcc ctgaccaagt caaatccatg cgggctgctc
     2341 ttgatctttt cggtcgtgag ttcggagacg tagccaccta ctcccaacat cagccggact
     2401 ccgattacct cgggaacttg ctccgtagta agacattcat cgcgcttgct gccttcgacc
     2461 aagaagcggt tgttggcgct ctcgcggctt acgttctgcc caagtttgag cagccgcgta
     2521 gtgagatcta tatctatgat ctcgcagtct ccggcgagca ccggaggcag ggcattgcca
     2581 ccgcgctcat caatctcctc aagcatgagg ccaacgcgct tggtgcttat gtgatctacg
     2641 tgcaagcaga ttacggtgac gatcccgcag tggctctcta tacaaagttg ggcatacggg
     2701 aagaagtgat gcactttgat atcgacccaa gtaccgccac ctaacaattc gttcaagccg
     2761 agatcggggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     2821 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     2881 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     2941 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3001 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3061 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3121 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3181 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3241 gaagaatagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3301 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3361 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3421 ctgacgctca gtggaacgaa aactccggat ctcctgttta aaccccgatt gagatatagt
     3481 ttttaattaa cccgtagtgg ctatggcagg gcttgccgcc ccgacgttgg ctgcgagccc
     3541 tgggccttca cccgaacttg ggggttgggg tggggaaaag gaagaaacgc gggcgtattg
     3601 gtcccaatgg ggtctcggtg gggtatcgac gtaaccccga ttgagatata gatttaaatc
     3661 tttagaccat agagcgttca cttgacagag tgccagccct gggaccgaac tcttctaggc
     3721 tcaagcagtg atcagcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
     3781 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
     3841 tgtttcaggt tcagggggag gtgctattcc tttgccctcg gacgagtgct ggggcgtcgg
     3901 tttccactat cggcgagtac ttctacacag ccatcggtcc agacggccgc gcttctgcgg
     3961 gcgatttgtg tacgcccgac agtcccggct ccggatcgga cgattgcgtc gcatcgaccc
     4021 tgcgcccaag ctgcatcatc gaaattgccg tcaaccaagc tctgatagag ttggtcaaga
     4081 ccaatgcgga gcatatacgc ccggagccgc ggcgatcctg caagctccgg atgcctccgc
     4141 tcgaagtagc gcgtctgctg ctccatacaa gccaaccacg gcctccagaa gaagatgttg
     4201 gcgacctcgt attgggaatc cccgaacatc gcctcgctcc agtcaatgac cgctgttatg
     4261 cggccattgt ccgtcaggac attgttggag ccgaaatccg cgtgcacgag gtgccggact
     4321 tcggggcagt cctcggccca aagcatcagc tcatcgagag cctgcgcgac ggacgcactg
     4381 acggtgtcgt ccatcacagt ttgccagtga tacacatggg gatcagcaat cgcgcatatg
     4441 aaatcacgcc atgtagtgta ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc
     4501 gtctggctaa gatcggccgc agcgatcgca tccatggcct ccgcgaccgg ctgcagaaca
     4561 gcgggcagtt cggtttcagg caggtcttgc aacgtgacac cctgtgcacg gcgggagatg
     4621 caataggtca ggctctcgct gaattcccca atgtcaagca cttccggaat cgggagcgcg
     4681 gccgatgcaa agtgccgata aacataacga tctttgtaga aaccatcggc gcagctattt
     4741 acccgcagga catatccacg ccctcctaca tcgaagctga aagcacgaga ttcttcgccc
     4801 tccgagagct gcatcaggtc ggagacgctg tcgaactttt cgatcagaaa cttctcgaca
     4861 gacgtcgcgg tgagttcagg ctttttcatg tatacgcgct agcggatctg acggttatat
     4921 ttcgataagc cagtaagcag tgggttctct agttagccag agctggggag agaggtcggt
     4981 gattcggtca acgagggagc cgactgccga cgtgcgctcc ggaggcttgc agaatgcgga
     5041 acaccgcgcg ggcaggaaca gggcccacac taccgcccca caccccgcct cccgcaccgc
     5101 cccttcccgg ccgctgctct cggcgcgccc tgctgagcag ccgctattgg ccacagccca
     5161 tcgcggtcgg cgcgctgcca ttgctccctg gcgctgtccg tctgcgaggg tactagtgag
     5221 acgtgcggct tccgtttgtc acgtccggca cgccgcgaac cgcaaggaac cttcccgact
     5281 taggggcgga gcaggaagcg tcgccggggg gcccacaagg gtagcggcga agatccgggt
     5341 gacgctgcga acggacgtga agaatgtgcg agacccaggg tcggcgccgc tgcgtttccc
     5401 ggaaccacgc ccagagcagc cgcgtccctg cgcaaaccca gggctgcctt ggaaaaggcg
     5461 caa
//
