LOCUS       sequence-299748-        4650 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid for Cre/lox-based recombinase-mediated cassette 
            exchange (RMCE), expressing one copy of etanercept.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4650)
  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 4650)
  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-ETN-lox2272
FEATURES             Location/Qualifiers
     source          1..4650
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(10..43)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     340..364
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      418..427
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(1906..1939)
                     /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(2414..2431)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2414..2430)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2529..2546)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2700..3288)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2780..2799)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3412..4203,4204..4272))
                     /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     4035..4054
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4273..4364)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4406..4425
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4431..4450
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 agtcggtgta taacttcgta tagcatacat tatacgaagt tatagtcggt gttgggactt
       61 tccaccttag atgacacagc aatcagattt gcttgcgtga gaagatatag gatgacacag
      121 caatctagac tgggactttc cactgatatt ttgcgcaatt gacctaatga cacagcaata
      181 gtatgtgggg cggggatcta actgggactt tccaaaggtc ttaccggaag ttgttagaat
      241 gacacagcaa tggattcata tcctgggact ttccagtata ctgcttgcgt gagaagatga
      301 tcatgggact ttccatgtac aaaaggtcta tataagcaga gctcgtttag tgaaccgtca
      361 gatcgcctag atacgccatc cacgctgttt tgacctccat agaagacaga cgtcatcgcc
      421 accatggcgc ccgtcgccgt ctgggccgcg ctggccgtcg gactggagct ctgggctgcg
      481 gcgcacgcct tgcccgccca ggtggcattt acaccctacg ccccggagcc cgggagcaca
      541 tgccggctca gagaatacta tgaccagaca gctcagatgt gctgcagcaa atgctcgccg
      601 ggccaacatg caaaagtctt ctgtaccaag acctcggaca ccgtgtgtga ctcctgtgag
      661 gacagcacat acacccagct ctggaactgg gttcccgagt gcttgagctg tggctcccgc
      721 tgtagctctg accaggtgga aactcaagcc tgcactcggg aacagaaccg catctgcacc
      781 tgcaggcccg gctggtactg cgcgctgagc aagcaggagg ggtgccggct gtgcgcgccg
      841 ctgcgcaagt gccgcccggg cttcggcgtg gccagaccag gaactgaaac atcagacgtg
      901 gtgtgcaagc cctgtgcccc ggggacgttc tccaacacga cttcatccac ggatatttgc
      961 aggccccacc agatctgtaa cgtggtggcc atccctggga atgcaagcat ggatgcagtc
     1021 tgcacgtcca cgtcccccac ccggagtatg gccccagggg cagtacactt accccagcca
     1081 gtgtccacac gatcccaaca cacgcagcca actccagaac ccagcactgc tccaagcacc
     1141 tccttcctgc tcccaatggg ccccagcccc ccagctgaag ggagcactgg cgacgagccc
     1201 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga
     1261 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
     1321 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg
     1381 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac
     1441 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag
     1501 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc
     1561 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag
     1621 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc
     1681 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg
     1741 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg
     1801 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca
     1861 cagaagagcc tctccctgtc tccgggtaaa tgataaaagc agcgtataac ttcgtatagg
     1921 atactttata cgaagttata cttgcgtagt gagtcgaata agggcgacac aaaatttatt
     1981 ctaaatgcat aataaatact gataacatct tatagtttgt attatatttt gtattatcgt
     2041 tgacatgtat aattttgata tcaaaaactg attttccctt tattattttc gagatttatt
     2101 ttcttaattc tctttaacaa actagaaata ttgtatatac aaaaaatcat aaataataga
     2161 tgaatagttt aattataggt gttcatcaat cgaaaaagca acgtatctta tttaaagtgc
     2221 gttgcttttt tctcatttat aaggttaaat aattctcata tatcaagcaa agtgacaggc
     2281 gcccttaaat attctgacaa atgctctttc cctaaactcc ccccataaaa aaacccgccg
     2341 aagcgggttt ttacgttatt tgcggattaa cgattactcg ttatcagaac cgcccagggg
     2401 gcccgagctt aagactggcc gtcgttttac aacacagaaa gagtttgtag aaacgcaaaa
     2461 aggccatccg tcaggggcct tctgcttagt ttgatgcctg gcagttccct actctcgcct
     2521 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     2581 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2641 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2701 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     2761 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     2821 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     2881 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     2941 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3001 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3061 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtgggct
     3121 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     3181 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3241 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3301 atcttttcta cggggtctga cgctcagtgg aacgacgcgc gcgtaactca cgttaaggga
     3361 ttttggtcat gagcttgcgc cgtcccgtca agtcagcgta atgctctgct tttaccaatg
     3421 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     3481 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagcgctgc
     3541 gatgataccg cgagaaccac gctcaccggc tccggattta tcagcaataa accagccagc
     3601 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     3661 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     3721 catcgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     3781 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     3841 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     3901 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     3961 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     4021 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     4081 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     4141 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     4201 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     4261 ttgaatactc atattcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     4321 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg tcagtgttac
     4381 aaccaattaa ccaattctga acattatcgc gagcccattt atacctgaat atggctcata
     4441 acaccccttg tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact
     4501 cagaagtgaa acgccgtagc gccgatggta gtgtggggac tccccatgcg agagtaggga
     4561 actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgcccgggc
     4621 taattatggg gtgtcgccct tattcgactc
//
