LOCUS       sequence-299742-        5503 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid for Cre/lox-based recombinase-mediated cassette 
            exchange (RMCE), expressing two copies of erythropoietin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5503)
  AUTHORS   Sergeeva D, Lee GM, Nielsen LK, Grav LM
  TITLE     Multi-copy targeted integration for accelerated development of 
            high-producing CHO cells.
  JOURNAL   ACS Synth Biol. 2020 Aug 24. doi: 10.1021/acssynbio.0c00322.
  PUBMED    32835482
REFERENCE   2  (bases 1 to 5503)
  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: "ACS Synth 
            Biol. 2020 Aug 24. doi: 10.1021/acssynbio.0c00322."
COMMENT     Sequence Label: 
            pJ204-loxP-100RPU.2-coEPO-BGHpA-cHS4-100RPU.2-coEPO-lox2272
FEATURES             Location/Qualifiers
     source          1..5503
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     134..153
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     159..178
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    complement(388..421)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      796..805
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1394..1618
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     regulatory      2540..2549
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3137..3170)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     primer_bind     complement(3645..3662)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3645..3661)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3760..3777)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3931..4519)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4011..4030)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4643..5434,5435..5503))
                     /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     5266..5285
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 attcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtc agtgttacaa ccaattaacc
      121 aattctgaac attatcgcga gcccatttat acctgaatat ggctcataac accccttgtt
      181 tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac
      241 gccgtagcgc cgatggtagt gtggggactc cccatgcgag agtagggaac tgccaggcat
      301 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gcccgggcta attatggggt
      361 gtcgccctta ttcgactcag tcggtgtata acttcgtata gcatacatta tacgaagtta
      421 tagtcggtgt tgggactttc caccttagat gacacagcaa tcagatttgc ttgcgtgaga
      481 agatatagga tgacacagca atctagactg ggactttcca ctgatatttt gcgcaattga
      541 cctaatgaca cagcaatagt atgtggggcg gggatctaac tgggactttc caaaggtctt
      601 accggaagtt gttagaatga cacagcaatg gattcatatc ctgggacttt ccagtatact
      661 gcttgcgtga gaagatgatc atgggacttt ccatgtacaa aaggtctata taagcagagc
      721 tcgtttagtg aaccgtcaga tcgcctagat acgccatcca cgctgttttg acctccatag
      781 aagacagacg tcatcgccac catgggagtg cacgagtgtc ctgcttggct gtggctgctg
      841 ctgtccctgc tgtctctgcc tctgggactg cctgtgctgg gcgctcctcc tagactgatc
      901 tgcgactccc gggtgctgga aagatacctg ctggaagcca aagaggccga gaacatcacc
      961 accggctgcg ccgagcactg ctccctgaac gagaatatca ccgtgcccga caccaaagtg
     1021 aacttctacg cctggaagcg gatggaagtg ggccagcagg ctgtggaagt gtggcaggga
     1081 ctggctctgc tgagcgaggc tgtgctgaga ggacaggccc tgctcgtgaa ctcctcccag
     1141 ccttgggaac ccctgcagct gcacgtggac aaggctgtgt ccggcctgag atccctgacc
     1201 accctgctga gagcactggg agcccagaaa gaggccatct ctccacctga cgccgcctct
     1261 gctgctcctc tgagaaccat caccgccgac accttcagaa agctgttccg ggtgtactcc
     1321 aacttcctgc ggggcaagct gaagctgtac accggcgagg cttgccggac cggcgataga
     1381 tgaaggtctg agtctgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     1441 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     1501 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     1561 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggat
     1621 aggctttatt taaatgggga cagccccccc ccaaagcccc cagggatgta attacgtccc
     1681 tcccccgcta gggggcagca gcgagccgcc cggggctccg ctccggtccg gcgctccccc
     1741 cgcatccccg agccggcagc gtgcggggac agcccgggca cggggaaggt ggcacgggat
     1801 cgctttcctc tgaacgcttc tcgctgctct ttgagcctgc agacacctgg ggggatacgg
     1861 ggaaaaagct ttaggctgag tgcacgatcg atacgtcgct gatatcattt aaatggggac
     1921 agcccccccc caaagccccc agggatgtaa ttacgtccct cccccgctag ggggcagcag
     1981 cgagccgccc ggggctccgc tccggtccgg cgctcccccc gcatccccga gccggcagcg
     2041 tgcggggaca gcccgggcac ggggaaggtg gcacgggatc gctttcctct gaacgcttct
     2101 cgctgctctt tgagcctgca gacacctggg gggatacggg gaaaaagctt taggctgagt
     2161 gcacgaacgt ggattgggac tttccacctt agatgacaca gcaatcagat ttgcttgcgt
     2221 gagaagatat aggatgacac agcaatctag actgggactt tccactgata ttttgcgcaa
     2281 ttgacctaat gacacagcaa tagtatgtgg ggcggggatc taactgggac tttccaaagg
     2341 tcttaccgga agttgttaga atgacacagc aatggattca tatcctggga ctttccagta
     2401 tactgcttgc gtgagaagat gatcatggga ctttccatgt acaaaaggtc tatataagca
     2461 gagctcgttt agtgaaccgt cagatcgcct agatacgcca tccacgctgt tttgacctcc
     2521 atagaagaca gacgtcatcg ccaccatggg agtgcacgag tgtcctgctt ggctgtggct
     2581 gctgctgtcc ctgctgtctc tgcctctggg actgcctgtg ctgggcgctc ctcctagact
     2641 gatctgcgac tcccgggtgc tggaaagata cctgctggaa gccaaagagg ccgagaacat
     2701 caccaccggc tgcgccgagc actgctccct gaacgagaat atcaccgtgc ccgacaccaa
     2761 agtgaacttc tacgcctgga agcggatgga agtgggccag caggctgtgg aagtgtggca
     2821 gggactggct ctgctgagcg aggctgtgct gagaggacag gccctgctcg tgaactcctc
     2881 ccagccttgg gaacccctgc agctgcacgt ggacaaggct gtgtccggcc tgagatccct
     2941 gaccaccctg ctgagagcac tgggagccca gaaagaggcc atctctccac ctgacgccgc
     3001 ctctgctgct cctctgagaa ccatcaccgc cgacaccttc agaaagctgt tccgggtgta
     3061 ctccaacttc ctgcggggca agctgaagct gtacaccggc gaggcttgcc ggaccggcga
     3121 tagatgaaag cagcgtataa cttcgtatag gatactttat acgaagttat acttgcgtag
     3181 tgagtcgaat aagggcgaca caaaatttat tctaaatgca taataaatac tgataacatc
     3241 ttatagtttg tattatattt tgtattatcg ttgacatgta taattttgat atcaaaaact
     3301 gattttccct ttattatttt cgagatttat tttcttaatt ctctttaaca aactagaaat
     3361 attgtatata caaaaaatca taaataatag atgaatagtt taattatagg tgttcatcaa
     3421 tcgaaaaagc aacgtatctt atttaaagtg cgttgctttt ttctcattta taaggttaaa
     3481 taattctcat atatcaagca aagtgacagg cgcccttaaa tattctgaca aatgctcttt
     3541 ccctaaactc cccccataaa aaaacccgcc gaagcgggtt tttacgttat ttgcggatta
     3601 acgattactc gttatcagaa ccgcccaggg ggcccgagct taagactggc cgtcgtttta
     3661 caacacagaa agagtttgta gaaacgcaaa aaggccatcc gtcaggggcc ttctgcttag
     3721 tttgatgcct ggcagttccc tactctcgcc ttccgcttcc tcgctcactg actcgctgcg
     3781 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     3841 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     3901 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     3961 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4021 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4081 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4141 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4201 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4261 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     4321 cggtgctaca gagttcttga agtggtgggc taactacggc tacactagaa gaacagtatt
     4381 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4441 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4501 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4561 gaacgacgcg cgcgtaactc acgttaaggg attttggtca tgagcttgcg ccgtcccgtc
     4621 aagtcagcgt aatgctctgc ttttaccaat gcttaatcag tgaggcacct atctcagcga
     4681 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4741 gggagggctt accatctggc cccagcgctg cgatgatacc gcgagaacca cgctcaccgg
     4801 ctccggattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4861 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4921 cgccagttaa tagtttgcgc aacgttgttg ccatcgctac aggcatcgtg gtgtcacgct
     4981 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5041 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5101 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5161 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5221 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5281 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5341 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5401 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5461 caaaaaaggg aataagggcg acacggaaat gttgaatact cat
//
