LOCUS       sequence-461257-        6298 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 mAGNLS-T2A-Hygro.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6298)
  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 6298)
  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 - mAGH
FEATURES             Location/Qualifiers
     source          1..6298
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      264..852
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     753..772
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(912..1445)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(1615..1620,1621..1637,1638..1643))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    complement(1671..1704)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    1775..1909
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1799..1818)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(1958..2557)
                     /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     2538..2557
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(2561..2617)
                     /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             complement(join(2618..2623,2624..2659,2660..2662,2663..2698,
                     2699..2701,2702..2737,2738..2743,2744..3361))
                     /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             complement(3362..3415)
                     /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"
     promoter        3656..4156
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4075..4093
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      4219..4228
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4225..4902
                     /codon_start=1
                     /product="humanized monomeric Azami-Green fluorescent 
                     protein"
                     /label=hmAzami-Green
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKIKLCMRGTVNGHNFVIEGEGKGNPYEGTQILDLNV
                     TEGAPLPFAYDILTTVFQYGNRAFTKYPADIQDYFKQTFPEGYHWERSMTYEDQGICTA
                     TSNISMRGDCFFYDIRFDGTNFPPNGPVMQKKTLKWEPSTEKMYVEDGVLKGDVNMRLL
                     LEGGGHYRCDFKTTYKAKKEVRLPDAHKIDHRIEILKHDKDYNKVKLYENAVARYSMLP
                     SQAK"
     CDS             4225..4902
                     /codon_start=1
                     /product="monomeric Azami-Green fluorescent protein 
                     (Karasawa et al., 2003)"
                     /label=mAzami-Green
                     /translation="MVSVIKPEMKIKLCMRGTVNGHNFVIEGEGKGNPYEGTQILDLNV
                     TEGAPLPFAYDILTTVFQYGNRAFTKYPADIQDYFKQTFPEGYHWERSMTYEDQGICTA
                     TSNISMRGDCFFYDIRFDGTNFPPNGPVMQKKTLKWEPSTEKMYVEDGVLKGDVNMRLL
                     LEGGGHYRCDFKTTYKAKKEVRLPDAHKIDHRIEILKHDKDYNKVKLYENAVARYSMLP
                     SQAK"
     CDS             4927..4947
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4951..4971
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4975..4995
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5005..5058
                     /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             5065..6090
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             5065..6090
                     /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"
     polyA_signal    6138..6219
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 aaatctatat ctcaatcggg gttacgtcga taccccaccg agaccccatt gggaccaata
       61 cgcccgcgtt tcttcctttt ccccacccca acccccaagt tcgggtgaag gcccagggct
      121 cgcagccaac gtcggggcgg caagccctgc catagccact acgggttaat taaaaactat
      181 atctcaatcg gggtttaaac aggagatccg gagttttcgt tccactgagc gtcagacccc
      241 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
      301 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
      361 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactat tcttctagtg
      421 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
      481 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
      541 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
      601 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
      661 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
      721 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
      781 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
      841 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcccc gatctcggct
      901 tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac ttcttcccgt
      961 atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc ttgcacgtag
     1021 atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag cgcggtggca
     1081 atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat ctcactacgc
     1141 ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc caacaaccgc ttcttggtcg
     1201 aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta atcggagtcc
     1261 ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag atcaagagca
     1321 gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat gcccatactt
     1381 gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt gctgctccat
     1441 aacatcaaac atcgacccac ggcgtaacgc gcttgctgct tggatgcccg aggcatagac
     1501 tgtacaaaaa aacagtcata acaagccatg aaaaccgcca ctgcgccgtt accaccgctg
     1561 cgttcggtca aggttctgga ccagttgcgt gagcgcatac gctacttgca ttacagttta
     1621 cgaaccgaac aggcttatgt caactgggtt cgtgccttcg ccgcggtacc ataacttcgt
     1681 atagcataca ttatacgaag ttatctgggt ttaaacgctc tatggtctaa agtttttatc
     1741 gaggctaact gaaacacgga acaagcagtg atcagatcca gacatgataa gatacattga
     1801 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     1861 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     1921 ttgcattcat tttatgtttc aggttcaggg ggaggtgtca ggcaccgggc ttgcgggtca
     1981 tgcaccaggt gcgcggtcct tcgggcacct cgacgtcggc ggtgacggtg aagccgagcc
     2041 gctcgtagaa ggggaggttg tggggcgcgg aggtctccag gaaggcgggc accccggcgc
     2101 gctcggccgc ctccactccg gggagcacga cggcgctgcc cagacccttg ccctggtggt
     2161 cgggcgagac gccgacggtg gccaggaacc acgcgggctc cttgggccgg tgcggcgcca
     2221 ggaggccttc catctgttgc tgcgcggcca gccgggaacc gctcaactcg gccatgcgcg
     2281 ggccgatctc ggcgaacacc gcccccgctt cgacgctctc cggcgtggtc cagaccgcca
     2341 ccgcggcgcc gtcgtccgcg acccacacct tgccgatgtc gagcccgacg cgcgtgagga
     2401 agagttcttg cagctcggtg acccgctcga tgtggcggtc cggatcgacg gtgtggcgcg
     2461 tggcggggta gtcggcgaac gcggcggcga gggtgcgtac ggccctgggg acgtcgtcgc
     2521 gggtggcgag gcgcaccgtg ggcttgtact cggtcatccc aggaccgggg ttttcttcca
     2581 cgtctcctgc ttgctttaac agagagaagt tcgtggcccc ggggagcatg tcaaggtcaa
     2641 aatcgtcaag agcgtcagca ggcagcatat caaggtcaaa gtcgtcaagg gcatcggctg
     2701 ggagcatgtc taagtcaaaa tcgtcaaggg cgtcggccgg cccgccgctt tcgcacttta
     2761 gctgtttctc caggccacat atgattagtt ccaggccgaa aaggaaggca ggttcggctc
     2821 cctgccggtc gaacagctca attgcttgtc tcagaagtgg gggcatagaa tcggtggtag
     2881 gtgtctctct ttcctctttt gctacttgat gctcctgttc ctccaatacg cagcccagtg
     2941 taaagtggcc cacggcggac agagcgtaca gtgcgttctc cagggagaag ccttgctgac
     3001 acaggaacgc gagctgattt tccagggttt cgtactgttt ctctgttggg cgggtgccga
     3061 gatgcacttt agccccgtcg cgatgtgaga ggagagcaca gcggaatgac ttggcgttgt
     3121 tccgcagaaa gtcttgccat gactcgcctt ccagggggca gaagtgggta tgatgcctgt
     3181 ccagcatctc gattggcagg gcatcgagca gggcccgctt gttcttcacg tgccagtaca
     3241 gggtaggctg ctcaactccc agcttttgag cgagtttcct tgtcgtcagg ccttcgatac
     3301 cgactccatt gagtaattcc agagcgccgt ttatgacttt gctcttgtcc agtctagaca
     3361 tggggccagg attctcctcg acgtcaccgc atgttagcag acttcctctg ccctctcccg
     3421 atccggcccc gaagcatttg cggtggacga tggaggggcc ggactcgtca tactcctgct
     3481 tgctgatcca catctgctgg aaggtggaca gcgaggccag gatggagccg ccgatccaca
     3541 cggagtactt gcgctcagga ggagcaatga tctgaggagg gaaggggaca ggcagtgagg
     3601 accctggatg tgacagctcc ccacacaccc tgtggtgtgt ggggagctgt ctcatttgcg
     3661 ccttttccaa ggcagccctg ggtttgcgca gggacgcggc tgctctgggc gtggttccgg
     3721 gaaacgcagc ggcgccgacc ctgggtctcg cacattcttc acgtccgttc gcagcgtcac
     3781 ccggatcttc gccgctaccc ttgtgggccc cccggcgacg cttcctgctc cgcccctaag
     3841 tcgggaaggt tccttgcggt tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct
     3901 cactagtacc ctcgcagacg gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg
     3961 ggctgtggcc aatagcggct gctcagcagg gcgcgccgag agcagcggcc gggaaggggc
     4021 ggtgcgggag gcggggtgtg gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc
     4081 cgcattctgc aagcctccgg agcgcacgtc ggcagtcggc tccctcgttg accgaatcac
     4141 cgacctctct ccccagctct ggctaactag agaacccact gcttactggc ttatcgaaat
     4201 ataaccgtca gatccgctgc caccatggtg agcgtgatca agcccgagat gaagatcaag
     4261 ctgtgcatga ggggcaccgt gaacggccac aacttcgtga tcgagggcga gggcaagggc
     4321 aacccctacg agggcaccca gatcctggac ctgaacgtga ccgagggcgc ccccctgccc
     4381 ttcgcctacg acatcctgac caccgtgttc cagtacggca acagggcctt caccaagtac
     4441 cccgccgaca tccaggacta cttcaagcag accttccccg agggctacca ctgggagagg
     4501 agcatgacct acgaggacca gggcatctgc accgccacca gcaacatcag catgaggggc
     4561 gactgcttct tctacgacat caggttcgac ggcaccaact tcccccccaa cggccccgtg
     4621 atgcagaaga agaccctgaa gtgggagccc agcaccgaga agatgtacgt ggaggacggc
     4681 gtgctgaagg gcgacgtgaa catgaggctg ctgctggagg gcggcggcca ctacaggtgc
     4741 gacttcaaga ccacctacaa ggccaagaag gaggtgaggc tgcccgacgc ccacaagatc
     4801 gaccacagga tcgagatcct gaagcacgac aaggactaca acaaggtgaa gctgtacgag
     4861 aacgccgtgg ccaggtactc catgctgccc agccaggcca agtccggact cagatctcga
     4921 gctgatccaa aaaagaagag aaaggtagat ccaaaaaaga agagaaaggt agatccaaaa
     4981 aagaagagaa aggtaggatc caccgagggc agaggaagtc tgctaacatg cggtgacgtc
     5041 gaggagaatc ctggccccgt atacatgaaa aagcctgaac tcaccgcgac gtctgtcgag
     5101 aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa
     5161 gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc
     5221 tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc
     5281 ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc
     5341 cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg
     5401 cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg
     5461 ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc
     5521 gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg
     5581 tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg
     5641 cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca
     5701 gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc
     5761 ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg
     5821 catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac
     5881 caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga
     5941 tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga
     6001 agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc
     6061 cccagcactc gtccgagggc aaaggaatag cacctccccc tgaacctgaa acaaaaatga
     6121 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     6181 gcatcacaaa tttcacaaat aaagcatttt tttcactgct gatcactgct tgagcctaga
     6241 agagttcggt cccagggctg gcactctgtc aagtgaacgc tctatggtct aaagattt
//
