LOCUS       sequence-438335-        5276 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3` circularization cassette.To induce Cre-mediated circularization 
            or inversion of a genomic region with concomitant GFP reconstitution
            and mScarlet expression from the chromosome.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5276)
  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 5276)
  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: 3` CC: Hygro – GFP-N – loxP – mScarlet
FEATURES             Location/Qualifiers
     source          1..5276
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        583..794
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             830..1852
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             830..1852
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1862..1915
                     /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"
     CDS             join(1925..1927,1928..2386)
                     /codon_start=1
                     /product="improved N-terminal fragment of mVenus for use in
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN155(I152L)
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMA"
     primer_bind     complement(1967..1988)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2228..2247)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    2430..2463
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2529..3221
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     complement(2659..2677)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2927..2946
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3225..3346
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3262..3281)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3316..3335
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(3374..3392)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    complement(3401..3500)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3517..3536)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3518..3536)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3541..3557)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3541..3557)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3659..3678)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3670..4479
                     /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
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGRPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3737..3756)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4626..5214
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5115..5134
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtgaattgg attggctccg gtgcccgtca gtgggcagag cgcacatcgc
      601 ccacagtccc cgagaagttg gggggagggg tcggcaattg atccggtgcc tagagaaggt
      661 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
      721 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
      781 ccgccagaac acaggaccgg ttctagagcg ctcgcccgcc ccacgagaga tgaaaaagcc
      841 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga
      901 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
      961 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     1021 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaatttag
     1081 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     1141 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc
     1201 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     1261 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     1321 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     1381 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     1441 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     1501 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     1561 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     1621 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     1681 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     1741 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     1801 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aaggcagtgg
     1861 agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaggggg
     1921 cggggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     1981 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     2041 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     2101 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     2161 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     2221 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     2281 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     2341 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca aggtaagttt
     2401 agtctttttg tcttttattt caggtcccga taacttcgta taatgtatgc tatacgaagt
     2461 tatggatccg gtggtggtgc aaatcaaaga actgctcctc agtggatgtt gcctttactt
     2521 ctagtgaggt gagcaagggc gaggcagtga tcaaggagtt catgcggttc aaggtgcaca
     2581 tggagggctc catgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct
     2641 acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttctcct
     2701 gggacatcct gtcccctcag ttcatgtacg gctccagggc cttcaccaag caccccgccg
     2761 acatccccga ctactataag cagtccttcc ccgagggctt caagtgggag cgcgtgatga
     2821 acttcgagga cggcggcgcc gtgaccgtga cccaggacac ctccctggag gacggcaccc
     2881 tgatctacaa ggtgaagctc cgcggcacca acttccctcc tgacggcccc gtaatgcaga
     2941 agaagacaat gggctgggaa gcgtccaccg agcggttgta ccccgaggac ggcgtgctga
     3001 agggcgacat taagatggcc ctgcgcctga aggacggcgg ccgctacctg gcggacttca
     3061 agaccaccta caaggccaag aagcccgtgc agatgcccgg cgcctacaac gtcgaccgca
     3121 agttggacat cacctcccac aacgaggact acaccgtggt ggaacagtac gaacgctccg
     3181 agggccgcca ctccaccggc ggcatggacg agctgtacaa gtagaacttg tttattgcag
     3241 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     3301 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggagct
     3361 ccagcttttg ttccctttag tgagggttaa tttcgagctt acccagcttt cttgtacaaa
     3421 gttggcatta taagaaagca ttgcttatca atttgttgca acgaacaggt cactatcagt
     3481 caaaataaaa tcattatttg ccatccagct gatatcccct atagtgagtc gtattacatg
     3541 gtcatagctg tttcctggca gctctggccc gtgtctcaaa atctctgatg ttacattgca
     3601 caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca
     3661 aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg
     3721 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     3781 atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     3841 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     3901 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact
     3961 gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat
     4021 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt
     4081 ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt
     4141 ttggttgatg cgagtgattt tgatgacgag cgtaatggcc ggcctgttga acaagtctgg
     4201 aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc
     4261 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga
     4321 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt
     4381 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat
     4441 aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg
     4501 taacactggc agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc
     4561 cttaacgtga gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     4621 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     4681 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4741 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     4801 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     4861 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4921 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4981 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     5041 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     5101 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     5161 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     5221 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgtt
//
