LOCUS       sequence-438333-        4323 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  5` circularization cassette.Used in combination with one of the 3' 
            circularization cassettes to induce Cre-mediated circularizazion or 
            inversion of a desired genomic region..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4323)
  AUTHORS   Pradella D, Zhang M, Gao R, Yao MA, Gluchowska KM, Cendon-Florez Y, 
            Mishra T, La Rocca G, Weigl M, Jiao Z, Nguyen HHM, Lisi M, Ozimek 
            MM, Mastroleo C, Chen K, Grimm F, Luebeck J, Zhang S, Zolli AA, Sun 
            EG, Dameracharla B, Zhao Z, Pritykin Y, Sigel C, Chang HY, Mischel 
            PS, Bafna V, Antonescu CR, Ventura A
  TITLE     Engineered extrachromosomal oncogene amplifications promote 
            tumorigenesis.
  JOURNAL   Nature. 2024 Dec 18. doi: 10.1038/s41586-024-08318-8.
  PUBMED    39695225
REFERENCE   2  (bases 1 to 4323)
  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: "Nature. 
            2024 Dec 18. doi: 10.1038/s41586-024-08318-8."
COMMENT     Sequence Label: 5` CC: Puro – loxP – GFP-C
FEATURES             Location/Qualifiers
     source          1..4323
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(877..904)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(996..1082)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     1131..1153
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1146..1162
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1178..1277
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1304..1814
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1733..1751
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1832..2428
                     /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(1832..1851)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2328..2348
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2438..2491
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     protein_bind    2537..2570
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2814..2835
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2881..3002
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2918..2937)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2972..2991
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(3030..3048)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    complement(3057..3156)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3173..3192)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3174..3192)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3197..3213)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3197..3213)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3315..3334)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3326..4135
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTV
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3393..3412)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(4282..4323,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
      781 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatac
      841 gcgtaccgct agccaggaag agtttgtaga aacgcaaaaa ggccatccgt caggatggcc
      901 ttctgcttag tttgatgcct ggcagtttat ggcgggcgtc ctgcccgcca ccctccgggc
      961 cgttgcttca caacgttcaa atccgctccc ggcggatttg tcctactcag gagagcgttc
     1021 accgacaaac aacagataaa acgaaaggcc cagtcttccg actgagcctt tcgttttatt
     1081 tgatgcctgg cagttcccta ctctcgcgtt aacgctagca tggatgtttt cccagtcacg
     1141 acgttgtaaa acgacggcca gtcttaagct cgggccccaa ataatgattt tattttgact
     1201 gatagtgacc tgttcgttgc aacaaattga ttagcaatgc ttttttataa tgccaacttt
     1261 gtacaaaaaa gcaggctaaa acgacggcca gtgaattggc cacggggttg gggttgcgcc
     1321 ttttccaagg cagccctggg tttgcgcagg gacgcggctg ctctgggcgt ggttccggga
     1381 aacgcagcgg cgccgaccct gggtctcgca cattcttcac gtccgttcgc agcgtcaccc
     1441 ggatcttcgc cgctaccctt gtgggccccc cggcgacgct tcctgctccg cccctaagtc
     1501 gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca
     1561 ctagtaccct cgcagacgga cagcgccagg gagcaatggc agcgcgccga ccgcgatggg
     1621 ctgtggccaa tagcggctgc tcagcagggc gcgccgagag cagcggccgg gaaggggcgg
     1681 tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg
     1741 cattctgcaa gcctccggag cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg
     1801 acctctctcc ccaggcgggc cccccctcac catgaccgag tacaagccca cggtgcgcct
     1861 cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta
     1921 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca
     1981 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     2041 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     2101 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     2161 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt
     2221 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     2281 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     2341 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     2401 ctggtgcatg acccgcaagc ccggtgccgg cagtggagag ggcagaggaa gtctgctaac
     2461 atgcggtgac gtcgaggaga atcctggccc agtgaagcgg taagtttagt ctttttgtct
     2521 tttatttcag gtcccgataa cttcgtataa tgtatgctat acgaagttat ggatccggtg
     2581 gtggtgcaaa tcaaagaact gctcctcagt ggatgttgcc tttacttcta gcagaagaac
     2641 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     2701 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     2761 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     2821 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtag
     2881 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     2941 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     3001 tatcatgtct ggagctccag cttttgttcc ctttagtgag ggttaatttc gagcttaccc
     3061 agctttcttg tacaaagttg gcattataag aaagcattgc ttatcaattt gttgcaacga
     3121 acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgata tcccctatag
     3181 tgagtcgtat tacatggtca tagctgtttc ctggcagctc tggcccgtgt ctcaaaatct
     3241 ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac tgtctgctta
     3301 cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg
     3361 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     3421 gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag agttgtttct
     3481 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     3541 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     3601 atggttactc accactgcga tccccggaaa aacagtattc caggtattag aagaatatcc
     3661 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     3721 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     3781 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     3841 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt
     3901 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     3961 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     4021 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     4081 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga
     4141 attggttaat tggttgtaac actggcagag cattacgctg acttgacggg acggcgcaag
     4201 ctcatgacca aaatccctta acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt
     4261 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4321 aac
//
