LOCUS       sequence-306203-        3757 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid for Cre/lox-based recombinase-mediated cassette 
            exchange (RMCE), expressing one copy of erythropoietin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3757)
  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 3757)
  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-lox2272
FEATURES             Location/Qualifiers
     source          1..3757
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(408..425)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(408..424)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(523..540)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(694..1282)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(774..793)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1404..2195,2196..2264))
                     /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     2027..2046
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2265..2356)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2398..2417
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2423..2442
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    complement(2652..2685)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2982..3006
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      3060..3069
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3657..3690)
                     /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)."
ORIGIN
        1 atcttatagt ttgtattata ttttgtatta tcgttgacat gtataatttt gatatcaaaa
       61 actgattttc cctttattat tttcgagatt tattttctta attctcttta acaaactaga
      121 aatattgtat atacaaaaaa tcataaataa tagatgaata gtttaattat aggtgttcat
      181 caatcgaaaa agcaacgtat cttatttaaa gtgcgttgct tttttctcat ttataaggtt
      241 aaataattct catatatcaa gcaaagtgac aggcgccctt aaatattctg acaaatgctc
      301 tttccctaaa ctccccccat aaaaaaaccc gccgaagcgg gtttttacgt tatttgcgga
      361 ttaacgatta ctcgttatca gaaccgccca gggggcccga gcttaagact ggccgtcgtt
      421 ttacaacaca gaaagagttt gtagaaacgc aaaaaggcca tccgtcaggg gccttctgct
      481 tagtttgatg cctggcagtt ccctactctc gccttccgct tcctcgctca ctgactcgct
      541 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
      601 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
      661 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
      721 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
      781 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
      841 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
      901 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
      961 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     1021 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     1081 aggcggtgct acagagttct tgaagtggtg ggctaactac ggctacacta gaagaacagt
     1141 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     1201 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     1261 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     1321 gtggaacgac gcgcgcgtaa ctcacgttaa gggattttgg tcatgagctt gcgccgtccc
     1381 gtcaagtcag cgtaatgctc tgcttaccaa tgcttaatca gtgaggcacc tatctcagcg
     1441 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     1501 cgggagggct taccatctgg ccccagcgct gcgatgatac cgcgagaacc acgctcaccg
     1561 gctccggatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     1621 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     1681 tcgccagtta atagtttgcg caacgttgtt gccatcgcta caggcatcgt ggtgtcacgc
     1741 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     1801 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     1861 aagttggccg cagtgttatc actcatggtt atggcagcgc tacataattc tcttactgtc
     1921 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     1981 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     2041 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     2101 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     2161 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     2221 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatattctt cctttttcaa
     2281 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     2341 tagaaaaata aacaaatagg ggtcagtgtt acaaccaatt aaccaattct gaacattatc
     2401 gcgagcccat ttatacctga atatggctca taacacccct tgtttgcctg gcggcagtag
     2461 cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg
     2521 tagtgtgggg actccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg
     2581 ctcagtcgaa agactgggcc tttcgcccgg gctaattatg gggtgtcgcc cttattcgac
     2641 tcagtcggtg tataacttcg tatagcatac attatacgaa gttatagtcg gtgttgggac
     2701 tttccacctt agatgacaca gcaatcagat ttgcttgcgt gagaagatat aggatgacac
     2761 agcaatctag actgggactt tccactgata ttttgcgcaa ttgacctaat gacacagcaa
     2821 tagtatgtgg ggcggggatc taactgggac tttccaaagg tcttaccgga agttgttaga
     2881 atgacacagc aatggattca tatcctggga ctttccagta tactgcttgc gtgagaagat
     2941 gatcatggga ctttccatgt acaaaaggtc tatataagca gagctcgttt agtgaaccgt
     3001 cagatcgcct agatacgcca tccacgctgt tttgacctcc atagaagaca gacgtcatcg
     3061 ccaccatggg agtgcacgag tgtcctgctt ggctgtggct gctgctgtcc ctgctgtctc
     3121 tgcctctggg actgcctgtg ctgggcgctc ctcctagact gatctgcgac tcccgggtgc
     3181 tggaaagata cctgctggaa gccaaagagg ccgagaacat caccaccggc tgcgccgagc
     3241 actgctccct gaacgagaat atcaccgtgc ccgacaccaa agtgaacttc tacgcctgga
     3301 agcggatgga agtgggccag caggctgtgg aagtgtggca gggactggct ctgctgagcg
     3361 aggctgtgct gagaggacag gccctgctcg tgaactcctc ccagccttgg gaacccctgc
     3421 agctgcacgt ggacaaggct gtgtccggcc tgagatccct gaccaccctg ctgagagcac
     3481 tgggagccca gaaagaggcc atctctccac ctgacgccgc ctctgctgct cctctgagaa
     3541 ccatcaccgc cgacaccttc agaaagctgt tccgggtgta ctccaacttc ctgcggggca
     3601 agctgaagct gtacaccggc gaggcttgcc ggaccggcga tagatgaaag cagcgtataa
     3661 cttcgtatag gatactttat acgaagttat acttgcgtag tgagtcgaat aagggcgaca
     3721 caaaatttat tctaaatgca taataaatac tgataac
//
