LOCUS       sequence-492680-        4878 bp    DNA     circular SYN 12-NOV-2025
DEFINITION  attP donor plasmid expressing puromycin resistance and mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4878)
  AUTHORS   Fanton A, Bartie LJ, Martins JQ, Tran VQ, Goudy L, Kernick C, 
            Durrant MG, Wei J, Armour-Garb Z, Pawluk A, Konermann S, Marson A, 
            Gilbert LA, Roth TL, Hsu PD
  TITLE     Site-specific DNA insertion into the human genome with engineered 
            recombinases.
  JOURNAL   Nat Biotechnol. 2025 Nov 6. doi: 10.1038/s41587-025-02895-3.
  PUBMED    41199024
REFERENCE   2  (bases 1 to 4878)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 12, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2025 Nov 6. doi: 10.1038/s41587-025-02895-3."
COMMENT     Sequence Label: pDonor_attP-Ef1a-Puro-P2A-mCherry
FEATURES             Location/Qualifiers
     source          1..4878
                     /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        2083..3261
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2314..3252
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3209..3229
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3269..3865
                     /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(3269..3288)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3765..3785
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3866..3922
                     /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             3929..4639
                     /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(4074..4092)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4342..4361
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(4664..4681)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4670..4877
                     /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 ttgggagccc taaactccca
     1981 cagataaaca gttaatggta atgaaataac attaactgtt tatctgtgtt taatgcctgc
     2041 cgcttcgtat tgttgccgcc gaatgcaacc tcgatcgagc cgggctccgg tgcccgtcag
     2101 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
     2161 accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
     2221 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
     2281 ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg
     2341 cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac ctggctgcag
     2401 tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc gaggccttgc
     2461 gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc
     2521 cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc
     2581 atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag tcttgtaaat
     2641 gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc
     2701 cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc accgagaatc
     2761 ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc gccgccgtgt
     2821 atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg agcggaaaga
     2881 tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag
     2941 cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca
     3001 tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc gagcttttgg
     3061 agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc ccacactgag
     3121 tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt ggaatttgcc
     3181 ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca aagttttttt
     3241 cttccatttc aggtgtcgtg aagccaccat gaccgagtac aagcccacgg tgcgcctcgc
     3301 cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
     3361 cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga
     3421 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     3481 cgccgtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     3541 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     3601 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggagtctc
     3661 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     3721 cgagcgcgcc ggggtgcccg ccttcctgga aacctccgcg ccccgcaacc tccccttcta
     3781 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     3841 gtgcatgacc cgcaagcccg gtgccgccac gaacttctct ctgttaaagc aagcaggtga
     3901 cgtggaagaa aaccccggtc ccggatccat ggtgagcaag ggcgaggagg ataacatggc
     3961 catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga
     4021 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     4081 gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat
     4141 gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc
     4201 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac
     4261 cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg
     4321 caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc
     4381 ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa
     4441 gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc
     4501 cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga
     4561 ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat
     4621 ggacgagctg tacaagtaac tcgagctaga gctcgctgat cagcctcgac tgtgccttct
     4681 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     4741 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     4801 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     4861 agcaggcatg ctggggac
//
