LOCUS       sequence-461254-        6326 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-mCherry-P2A-puro and mTagBFPnls-T2A-Hygro.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6326)
  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 6326)
  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 ActBT2AmcherryP2AP -TagBFPH
FEATURES             Location/Qualifiers
     source          1..6326
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(40..1065)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(40..1065)
                     /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"
     CDS             complement(1072..1125)
                     /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             complement(1135..1155)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1159..1179)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1183..1203)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1228..1926)
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     promoter        complement(2011..2511)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2074..2092)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2752..2805
                     /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"
     regulatory      2812..2821
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2818..3525
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2963..2981)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3231..3250
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3541..3597
                     /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             3601..4200
                     /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(3601..3620)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     polyA_signal    4249..4383
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4340..4359
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    4454..4487
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        join(4515..4520,4521..4537,4538..4543)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             4713..5246
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     rep_origin      complement(5306..5894)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5386..5405)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    6237..6318
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 caattgcatt catttttgtt tcaggttcag ggggaggtgc tattcctttg ccctcggacg
       61 agtgctgggg cgtcggtttc cactatcggc gagtacttct acacagccat cggtccagac
      121 ggccgcgctt ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg atcggacgat
      181 tgcgtcgcat cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg
      241 atagagttgg tcaagaccaa tgcggagcat atacgcccgg agccgcggcg atcctgcaag
      301 ctccggatgc ctccgctcga agtagcgcgt ctgctgctcc atacaagcca accacggcct
      361 ccagaagaag atgttggcga cctcgtattg ggaatccccg aacatcgcct cgctccagtc
      421 aatgaccgct gttatgcggc cattgtccgt caggacattg ttggagccga aatccgcgtg
      481 cacgaggtgc cggacttcgg ggcagtcctc ggcccaaagc atcagctcat cgagagcctg
      541 cgcgacggac gcactgacgg tgtcgtccat cacagtttgc cagtgataca catggggatc
      601 agcaatcgcg catatgaaat cacgccatgt agtgtattga ccgattcctt gcggtccgaa
      661 tgggccgaac ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca tggcctccgc
      721 gaccggctgc agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg
      781 tgcacggcgg gagatgcaat aggtcaggct ctcgctgaat tccccaatgt caagcacttc
      841 cggaatcggg agcgcggccg atgcaaagtg ccgataaaca taacgatctt tgtagaaacc
      901 atcggcgcag ctatttaccc gcaggacata tccacgccct cctacatcga agctgaaagc
      961 acgagattct tcgccctccg agagctgcat caggtcggag acgctgtcga acttttcgat
     1021 cagaaacttc tcgacagacg tcgcggtgag ttcaggcttt ttcatgtata cggggccagg
     1081 attctcctcg acgtcaccgc atgttagcag acttcctctg ccctcggtgg atcctacctt
     1141 tctcttcttt tttggatcta cctttctctt cttttttgga tctacctttc tcttcttttt
     1201 tggatcagct cgagatctga gtccggaatt aagcttgtgc cccagtttgc tagggaggtc
     1261 gcagtatctg gccactgcca cctcgtgctg ctcgacgtag gtctcgttgt tggcctcctt
     1321 gattctttcc agtctgtagt ccacatagta gacgccaggc atcttgaggt tcttagcggg
     1381 tttcttggat ctatatgtgg tcttgatgtt tgcgatcaga tggctcccgc ccacgagctt
     1441 cagggccatg tcgtttctgc cttccaggcc gccgtcagcg gggtacagcg tctcggtgaa
     1501 ggcctcccag ccgagtgttt tcttctgcat cacagggccg ttggatgtga agttcacccc
     1561 tctgatcttg acgttgtaga tgaggcagcc gtcctggagg ctggtgtcct gggtagcggt
     1621 cagcacgccc ccgtcttcgt atgtggtgac tctctcccat gtgaagccct cagggaagga
     1681 ctgcttgaag aagtcgggga tgccctgggt gtggttgatg aaggtcttgc tgccgtagag
     1741 gaagctagta gccaggatgt cgaaggcgaa ggggagaggg ccgccctcga ccaccttgat
     1801 tctcatggtc tgggtgccct cgtagggctt gccttcgccc tcggatgtgc acttgaagtg
     1861 atggttgtcc acggtgccct ccatgtacag cttcatgtgc atgttctcct taatcagctc
     1921 gctcatggtg gcgaccggta tacgcgctag cggatctgac ggttatattt cgataagcca
     1981 gtaagcagtg ggttctctag ttagccagag ctggggagag aggtcggtga ttcggtcaac
     2041 gagggagccg actgccgacg tgcgctccgg aggcttgcag aatgcggaac accgcgcggg
     2101 caggaacagg gcccacacta ccgccccaca ccccgcctcc cgcaccgccc cttcccggcc
     2161 gctgctctcg gcgcgccctg ctgagcagcc gctattggcc acagcccatc gcggtcggcg
     2221 cgctgccatt gctccctggc gctgtccgtc tgcgagggta ctagtgagac gtgcggcttc
     2281 cgtttgtcac gtccggcacg ccgcgaaccg caaggaacct tcccgactta ggggcggagc
     2341 aggaagcgtc gccggggggc ccacaagggt agcggcgaag atccgggtga cgctgcgaac
     2401 ggacgtgaag aatgtgcgag acccagggtc ggcgccgctg cgtttcccgg aaccacgccc
     2461 agagcagccg cgtccctgcg caaacccagg gctgccttgg aaaaggcgca aatgagacag
     2521 ctccccacac accacagggt gtgtggggag ctgtcacatc cagggtcctc actgcctgtc
     2581 cccttccctc ctcagatcat tgctcctcct gagcgcaagt actccgtgtg gatcggcggc
     2641 tccatcctgg cctcgctgtc caccttccag cagatgtgga tcagcaagca ggagtatgac
     2701 gagtccggcc cctccatcgt ccaccgcaaa tgcttcgggg ccggatcggg agagggcaga
     2761 ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gccccggggt cgccaccatg
     2821 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
     2881 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     2941 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     3001 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     3061 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     3121 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     3181 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     3241 cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     3301 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
     3361 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     3421 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     3481 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagggggc cggatcggga
     3541 gccacgaact tctctctgtt aaagcaagca ggagacgtgg aagaaaaccc cggtcccggg
     3601 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     3661 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     3721 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     3781 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     3841 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     3901 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     3961 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
     4021 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     4081 gagacctccg cgccccacaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     4141 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga
     4201 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     4261 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4321 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     4381 atctgatcac tgcttgttcc gtgtttcagt tagcctcgat aaaaacttta gaccatagag
     4441 cgtttaaacc cagataactt cgtataatgt atgctatacg aagttatggt accgcggcga
     4501 aggcacgaac ccagttgaca taagcctgtt cggttcgtaa actgtaatgc aagtagcgta
     4561 tgcgctcacg caactggtcc agaaccttga ccgaacgcag cggtggtaac ggcgcagtgg
     4621 cggttttcat ggcttgttat gactgttttt ttgtacagtc tatgcctcgg gcatccaagc
     4681 agcaagcgcg ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc
     4741 agcagggcag tcgccctaaa acaaagttag gtggctcaag tatgggcatc attcgcacat
     4801 gtaggctcgg ccctgaccaa gtcaaatcca tgcgggctgc tcttgatctt ttcggtcgtg
     4861 agttcggaga cgtagccacc tactcccaac atcagccgga ctccgattac ctcgggaact
     4921 tgctccgtag taagacattc atcgcgcttg ctgccttcga ccaagaagcg gttgttggcg
     4981 ctctcgcggc ttacgttctg cccaagtttg agcagccgcg tagtgagatc tatatctatg
     5041 atctcgcagt ctccggcgag caccggaggc agggcattgc caccgcgctc atcaatctcc
     5101 tcaagcatga ggccaacgcg cttggtgctt atgtgatcta cgtgcaagca gattacggtg
     5161 acgatcccgc agtggctctc tatacaaagt tgggcatacg ggaagaagtg atgcactttg
     5221 atatcgaccc aagtaccgcc acctaacaat tcgttcaagc cgagatcggg gccaggaacc
     5281 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5341 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5401 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5461 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5521 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5581 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5641 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5701 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaata gtatttggta
     5761 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5821 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5881 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5941 aaaactccgg atctcctgtt taaaccccga ttgagatata gtttttaatt aacccgtagt
     6001 ggctatggca gggcttgccg ccccgacgtt ggctgcgagc cctgggcctt cacccgaact
     6061 tgggggttgg ggtggggaaa aggaagaaac gcgggcgtat tggtcccaat ggggtctcgg
     6121 tggggtatcg acgtaacccc gattgagata tagatttaaa tctttagacc atagagcgtt
     6181 cacttgacag agtgccagcc ctgggaccga actcttctag gctcaagcag tgatcagcag
     6241 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
     6301 agctgcaata aacaagttaa caacaa
//
