LOCUS       sequence-378313-        4923 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dn29 Donor with promoter for mcherry and puro expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4923)
  AUTHORS   Durrant MG, Fanton A, Tycko J, Hinks M, Chandrasekaran SS, Perry NT,
            Schaepe J, Du PP, Lotfy P, Bassik MC, Bintu L, Bhatt AS, Hsu PD
  TITLE     Systematic discovery of recombinases for efficient integration of 
            large DNA sequences into the human genome.
  JOURNAL   Nat Biotechnol. 2022 Oct 10. pii: 10.1038/s41587-022-01494-w. doi: 
            10.1038/s41587-022-01494-w.
  PUBMED    36217031
REFERENCE   2  (bases 1 to 4923)
  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: "Nat 
            Biotechnol. 2022 Oct 10. pii: 10.1038/s41587-022-01494-w. doi: 
            10.1038/s41587-022-01494-w."
COMMENT     Sequence Label: pAF0692 Ef1a-Dn29_attD-Puro-p2a-mCherry
FEATURES             Location/Qualifiers
     source          1..4923
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        32..136
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(137..205,206..997)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(355..374)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1168..1756
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1657..1676
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1910..1927
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        2128..3306
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2359..3297
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3254..3274
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3314..3910
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3314..3333)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3810..3830
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3911..3967
                     /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             3974..4684
                     /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(4119..4137)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4387..4406
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(4709..4726)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4715..4922
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
       61 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
      121 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
      181 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
      241 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
      301 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
      361 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
      421 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
      481 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
      541 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
      601 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
      661 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
      721 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
      781 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
      841 cggtgagcgt ggctctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
      901 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
      961 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     1021 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     1081 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     1141 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     1201 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     1261 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     1321 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     1381 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     1441 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     1501 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     1561 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     1621 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     1681 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     1741 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     1801 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     1861 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     1921 gagcgaggaa gcggaagagc gagcttcgca cgattgcacc ttgggagctg attttacgct
     1981 ggtgctatat cctaaactcc cacagataaa cagttaatgg taatgaaata acattaactg
     2041 tttatctgtg tttaatgcct taacttaatc tagtaggagg ggccnngtat staakncnag
     2101 ccgccgaatg caacctcgat cgagccgggc tccggtgccc gtcagtgggc agagcgcaca
     2161 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga
     2221 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
     2281 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
     2341 tttgccgcca gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg
     2401 ggttatggcc cttgcgtgcc ttgaattact tccacctggc tgcagtacgt gattcttgat
     2461 cccgagcttc gggttggaag tgggtgggag agttcgaggc cttgcgctta aggagcccct
     2521 tcgcctcgtg cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg
     2581 tggcaccttc gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga
     2641 tgacctgctg cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaagatctg
     2701 cacactggta tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc
     2761 acatgttcgg cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct
     2821 caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg
     2881 gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc
     2941 cctgctgcag ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca
     3001 cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag
     3061 taccgggcgc cgtccaggca cctcgattag ttctcgagct tttggagtac gtcgtcttta
     3121 ggttgggggg aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa
     3181 gttaggccag cttggcactt gatgtaattc tccttggaat ttgccctttt tgagtttgga
     3241 tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg
     3301 tcgtgaagcc accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     3361 ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     3421 cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     3481 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgccg tggcggtctg
     3541 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     3601 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     3661 ccggcccaag gagcccgcgt ggttcctggc caccgtcgga gtctcgcccg accaccaggg
     3721 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     3781 gcccgccttc ctggaaacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     3841 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     3901 gcccggtgcc gccacgaact tctctctgtt aaagcaagca ggtgacgtgg aagaaaaccc
     3961 cggtcccgga tccatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
     4021 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
     4081 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     4141 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
     4201 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
     4261 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
     4321 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
     4381 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta
     4441 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     4501 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     4561 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt
     4621 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     4681 gtaactcgag ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     4741 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     4801 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     4861 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     4921 gac
//
