LOCUS       sequence-438331-        5654 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3` circularization cassette to induce Cre-mediated circularization 
            of a genomic region flanked by loxP sites with H2B-GFP 
            reconstitution and mScarlet expression from the chromosome.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5654)
  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 5654)
  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 – H2B-GFP-N – loxP – mScarlet
FEATURES             Location/Qualifiers
     source          1..5654
                     /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     1145..1162
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1146..1162
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1192..1403
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             1439..2461
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1439..2461
                     /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             2471..2524
                     /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             2525..2902
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     CDS             join(2912..2914,2915..3373)
                     /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(2954..2975)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3215..3234)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    3417..3450
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3516..4208
                     /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(3646..3664)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3914..3933
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4212..4333
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4249..4268)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4303..4322
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(4361..4379)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    complement(4388..4487)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4504..4523)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4505..4523)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4528..4544)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4528..4544)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4646..4665)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4657..5466
                     /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(4724..4743)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(5613..5654,1..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 gtgaattgga ttggctccgg tgcccgtcag tgggcagagc
     1201 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga tccggtgcct
     1261 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc
     1321 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca
     1381 acgggtttgc cgccagaaca caggaccggt tctagagcgc tcgcccgccc cacgagagat
     1441 gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag
     1501 cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt
     1561 aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg
     1621 ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg
     1681 ggaatttagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca
     1741 agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc
     1801 gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat
     1861 cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca
     1921 ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct
     1981 gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc
     2041 caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat
     2101 gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg
     2161 tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg
     2221 gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg
     2281 caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc
     2341 cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg
     2401 tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga
     2461 aggcagtgga gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg
     2521 cccaatgcca gagccagcga agtctgctcc cgccccgaaa aagggctcca agaaggcggt
     2581 gactaaggcg cagaagaaag gcggcaagaa gcgcaagcgc agccgcaagg agagctattc
     2641 catctatgtg tacaaggttc tgaagcaggt ccaccctgac accggcattt cgtccaaggc
     2701 catgggcatc atgaattcgt ttgtgaacga cattttcgag cgcatcgcag gtgaggcttc
     2761 ccgcctggcg cattacaaca agcgctcgac catcacctcc agggagatcc agacggccgt
     2821 gcgcctgctg ctgcctgggg agttggccaa gcacgccgtg tccgagggta ctaaggccat
     2881 caccaagtac accagcgcta aggggggcgg ggtgagcaag ggcgaggagc tgttcaccgg
     2941 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     3001 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     3061 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     3121 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     3181 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     3241 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     3301 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     3361 ctatatcatg gccgacaagg taagtttagt ctttttgtct tttatttcag gtcccgataa
     3421 cttcgtataa tgtatgctat acgaagttat ggatccggtg gtggtgcaaa tcaaagaact
     3481 gctcctcagt ggatgttgcc tttacttcta gtgaggtgag caagggcgag gcagtgatca
     3541 aggagttcat gcggttcaag gtgcacatgg agggctccat gaacggccac gagttcgaga
     3601 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
     3661 ccaagggtgg ccccctgccc ttctcctggg acatcctgtc ccctcagttc atgtacggct
     3721 ccagggcctt caccaagcac cccgccgaca tccccgacta ctataagcag tccttccccg
     3781 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgccgtg accgtgaccc
     3841 aggacacctc cctggaggac ggcaccctga tctacaaggt gaagctccgc ggcaccaact
     3901 tccctcctga cggccccgta atgcagaaga agacaatggg ctgggaagcg tccaccgagc
     3961 ggttgtaccc cgaggacggc gtgctgaagg gcgacattaa gatggccctg cgcctgaagg
     4021 acggcggccg ctacctggcg gacttcaaga ccacctacaa ggccaagaag cccgtgcaga
     4081 tgcccggcgc ctacaacgtc gaccgcaagt tggacatcac ctcccacaac gaggactaca
     4141 ccgtggtgga acagtacgaa cgctccgagg gccgccactc caccggcggc atggacgagc
     4201 tgtacaagta gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4261 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4321 tcaatgtatc ttatcatgtc tggagctcca gcttttgttc cctttagtga gggttaattt
     4381 cgagcttacc cagctttctt gtacaaagtt ggcattataa gaaagcattg cttatcaatt
     4441 tgttgcaacg aacaggtcac tatcagtcaa aataaaatca ttatttgcca tccagctgat
     4501 atcccctata gtgagtcgta ttacatggtc atagctgttt cctggcagct ctggcccgtg
     4561 tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg aacaataaaa
     4621 ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg
     4681 tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc
     4741 gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca
     4801 gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc
     4861 agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact
     4921 cctgatgatg catggttact caccactgcg atccccggaa aaacagcatt ccaggtatta
     4981 gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg
     5041 ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct
     5101 caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt
     5161 aatggccggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg
     5221 gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa
     5281 ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc
     5341 atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa
     5401 tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt
     5461 ttctaatcag aattggttaa ttggttgtaa cactggcaga gcattacgct gacttgacgg
     5521 gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg
     5581 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     5641 tgctgcttgc aaac
//
