LOCUS       sequence-308566-        7283 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid for Cre/lox-based recombinase-mediated cassette 
            exchange (RMCE), expressing two copies of etanercept.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7283)
  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 7283)
  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-BGHpA-cHS4-100RPU.2-ETN-lox2272
FEATURES             Location/Qualifiers
     source          1..7283
                     /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    2285..2509
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     regulatory      3431..3440
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(4919..4952)
                     /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(5427..5444)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5427..5443)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5542..5559)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5713..6301)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5793..5812)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6423..7214,7215..7283))
                     /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     7046..7065
                     /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 catggcgccc gtcgccgtct gggccgcgct ggccgtcgga
      841 ctggagctct gggctgcggc gcacgccttg cccgcccagg tggcatttac accctacgcc
      901 ccggagcccg ggagcacatg ccggctcaga gaatactatg accagacagc tcagatgtgc
      961 tgcagcaaat gctcgccggg ccaacatgca aaagtcttct gtaccaagac ctcggacacc
     1021 gtgtgtgact cctgtgagga cagcacatac acccagctct ggaactgggt tcccgagtgc
     1081 ttgagctgtg gctcccgctg tagctctgac caggtggaaa ctcaagcctg cactcgggaa
     1141 cagaaccgca tctgcacctg caggcccggc tggtactgcg cgctgagcaa gcaggagggg
     1201 tgccggctgt gcgcgccgct gcgcaagtgc cgcccgggct tcggcgtggc cagaccagga
     1261 actgaaacat cagacgtggt gtgcaagccc tgtgccccgg ggacgttctc caacacgact
     1321 tcatccacgg atatttgcag gccccaccag atctgtaacg tggtggccat ccctgggaat
     1381 gcaagcatgg atgcagtctg cacgtccacg tcccccaccc ggagtatggc cccaggggca
     1441 gtacacttac cccagccagt gtccacacga tcccaacaca cgcagccaac tccagaaccc
     1501 agcactgctc caagcacctc cttcctgctc ccaatgggcc ccagcccccc agctgaaggg
     1561 agcactggcg acgagcccaa atcttgtgac aaaactcaca catgcccacc gtgcccagca
     1621 cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc
     1681 atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct
     1741 gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg
     1801 cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag
     1861 gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc
     1921 atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg
     1981 cccccatccc gggaggagat gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc
     2041 ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac
     2101 aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc
     2161 gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct
     2221 ctgcacaacc actacacaca gaagagcctc tccctgtctc cgggtaaatg ataaaggtct
     2281 gagtctgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     2341 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     2401 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     2461 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatgga taggctttat
     2521 ttaaatgggg acagcccccc cccaaagccc ccagggatgt aattacgtcc ctcccccgct
     2581 agggggcagc agcgagccgc ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc
     2641 gagccggcag cgtgcgggga cagcccgggc acggggaagg tggcacggga tcgctttcct
     2701 ctgaacgctt ctcgctgctc tttgagcctg cagacacctg gggggatacg gggaaaaagc
     2761 tttaggctga gtgcacgatc gatacgtcgc tgatatcatt taaatgggga cagccccccc
     2821 ccaaagcccc cagggatgta attacgtccc tcccccgcta gggggcagca gcgagccgcc
     2881 cggggctccg ctccggtccg gcgctccccc cgcatccccg agccggcagc gtgcggggac
     2941 agcccgggca cggggaaggt ggcacgggat cgctttcctc tgaacgcttc tcgctgctct
     3001 ttgagcctgc agacacctgg ggggatacgg ggaaaaagct ttaggctgag tgcacgaacg
     3061 tggattggga ctttccacct tagatgacac agcaatcaga tttgcttgcg tgagaagata
     3121 taggatgaca cagcaatcta gactgggact ttccactgat attttgcgca attgacctaa
     3181 tgacacagca atagtatgtg gggcggggat ctaactggga ctttccaaag gtcttaccgg
     3241 aagttgttag aatgacacag caatggattc atatcctggg actttccagt atactgcttg
     3301 cgtgagaaga tgatcatggg actttccatg tacaaaaggt ctatataagc agagctcgtt
     3361 tagtgaaccg tcagatcgcc tagatacgcc atccacgctg ttttgacctc catagaagac
     3421 agacgtcatc gccaccatgg cgcccgtcgc cgtctgggcc gcgctggccg tcggactgga
     3481 gctctgggct gcggcgcacg ccttgcccgc ccaggtggca tttacaccct acgccccgga
     3541 gcccgggagc acatgccggc tcagagaata ctatgaccag acagctcaga tgtgctgcag
     3601 caaatgctcg ccgggccaac atgcaaaagt cttctgtacc aagacctcgg acaccgtgtg
     3661 tgactcctgt gaggacagca catacaccca gctctggaac tgggttcccg agtgcttgag
     3721 ctgtggctcc cgctgtagct ctgaccaggt ggaaactcaa gcctgcactc gggaacagaa
     3781 ccgcatctgc acctgcaggc ccggctggta ctgcgcgctg agcaagcagg aggggtgccg
     3841 gctgtgcgcg ccgctgcgca agtgccgccc gggcttcggc gtggccagac caggaactga
     3901 aacatcagac gtggtgtgca agccctgtgc cccggggacg ttctccaaca cgacttcatc
     3961 cacggatatt tgcaggcccc accagatctg taacgtggtg gccatccctg ggaatgcaag
     4021 catggatgca gtctgcacgt ccacgtcccc cacccggagt atggccccag gggcagtaca
     4081 cttaccccag ccagtgtcca cacgatccca acacacgcag ccaactccag aacccagcac
     4141 tgctccaagc acctccttcc tgctcccaat gggccccagc cccccagctg aagggagcac
     4201 tggcgacgag cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga
     4261 actcctgggg ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat
     4321 ctcccggacc cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt
     4381 caagttcaac tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga
     4441 ggagcagtac aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg
     4501 gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga
     4561 gaaaaccatc tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc
     4621 atcccgggag gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta
     4681 tcccagcgac atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac
     4741 cacgcctccc gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga
     4801 caagagcagg tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca
     4861 caaccactac acacagaaga gcctctccct gtctccgggt aaatgataaa agcagcgtat
     4921 aacttcgtat aggatacttt atacgaagtt atacttgcgt agtgagtcga ataagggcga
     4981 cacaaaattt attctaaatg cataataaat actgataaca tcttatagtt tgtattatat
     5041 tttgtattat cgttgacatg tataattttg atatcaaaaa ctgattttcc ctttattatt
     5101 ttcgagattt attttcttaa ttctctttaa caaactagaa atattgtata tacaaaaaat
     5161 cataaataat agatgaatag tttaattata ggtgttcatc aatcgaaaaa gcaacgtatc
     5221 ttatttaaag tgcgttgctt ttttctcatt tataaggtta aataattctc atatatcaag
     5281 caaagtgaca ggcgccctta aatattctga caaatgctct ttccctaaac tccccccata
     5341 aaaaaacccg ccgaagcggg tttttacgtt atttgcggat taacgattac tcgttatcag
     5401 aaccgcccag ggggcccgag cttaagactg gccgtcgttt tacaacacag aaagagtttg
     5461 tagaaacgca aaaaggccat ccgtcagggg ccttctgctt agtttgatgc ctggcagttc
     5521 cctactctcg ccttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5581 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5641 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5701 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5761 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5821 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     5881 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     5941 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6001 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6061 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6121 gaagtggtgg gctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     6181 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6241 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6301 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgacg cgcgcgtaac
     6361 tcacgttaag ggattttggt catgagcttg cgccgtcccg tcaagtcagc gtaatgctct
     6421 gcttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6481 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6541 cccagcgctg cgatgatacc gcgagaacca cgctcaccgg ctccggattt atcagcaata
     6601 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6661 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6721 aacgttgttg ccatcgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6781 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6841 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6901 ctcatggtta tggcagcgct acataattct cttactgtca tgccatccgt aagatgcttt
     6961 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     7021 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     7081 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7141 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7201 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7261 acacggaaat gttgaatact cat
//
