LOCUS       sequence-382886-        5302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bicistronic DTR-EGFP fusion and Puromycin resistance for 
            positive/negative selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5302)
  AUTHORS   Barriga FM, Tsanov KM, Ho YJ, Sohail N, Zhang A, Baslan T, Wuest AN,
            Del Priore I, Meskauskaite B, Livshits G, Alonso-Curbelo D, Simon J,
            Chaves-Perez A, Bar-Sagi D, Iacobuzio-Donahue CA, Notta F, Chaligne 
            R, Sharma R, Pe'er D, Lowe SW
  TITLE     MACHETE identifies interferon-encompassing chromosome 9p21.3 
            deletions as mediators of immune evasion and metastasis.
  JOURNAL   Nat Cancer. 2022 Nov;3(11):1367-1385. doi: 
            10.1038/s43018-022-00443-5. Epub 2022 Nov 7.
  PUBMED    36344707
REFERENCE   2  (bases 1 to 5302)
  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"; doi: 
            "10.1038/s43018-022-00443-5"; journalName: "Nat Cancer"; date: 
            "2022-11"; volume: "3"; issue: "11"; pages: "1367-1385"
COMMENT     Sequence Label: pFB2D
FEATURES             Location/Qualifiers
     source          1..5302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     35..52
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    169..190
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(205..210,211..228,229..235)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    243..259
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     248..270
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     267..283
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     267..283
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        326..537
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             join(1167..1169,1170..1172,1173..1883)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1167..1169,1170..1172,1173..1883)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1212..1233)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1473..1492)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1820..1841
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1893..1946
                     /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             1947..2546
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1947..1966)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(2557..2574)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2563..2787
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2820..2837)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2820..2836)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2829..2851)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2977..3432)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3114..3135)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3326..3345
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3459..3563
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3564..3632,3633..4424)
                     /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     complement(3782..3801)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4595..5183
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5084..5103
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
       61 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
      121 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
      181 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
      241 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgagg cgcgcccagg
      301 agctattaat tcgcggaggg aattcgggca gagcgcacat cgcccacagt ccccgagaag
      361 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg
      421 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
      481 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacagcca
      541 ccatgaagct gctgccgtcg gtagtgctga agctctttct ggctgcagtt ctctcggcac
      601 tggtgactgg cgagagcctg gagcggcttc ggagagggct agctgctgga accagcaacc
      661 cggaccctcc cactgtatcc acggaccagc tgctacccct aggaggcggc cgggaccgga
      721 aagtccgtga cttgcaagag gcagatctgg accttttgag agtcacttta tcctccaagc
      781 cacaagcact ggccacacca aacaaggagg agcacgggaa aagaaagaag aaaggcaagg
      841 ggctagggaa gaagagggac ccatgtcttc ggaaatacaa ggacttctgc atccatggag
      901 aatgcaaata tgtgaaggag ctccgggctc cctcctgcat ctgccacccg ggttaccatg
      961 gagagaggtg tcatgggctg agcctcccag tggaaaatcg cttatatacc tatgaccaca
     1021 caaccatcct ggccgtggtg gctgtggtgc tgtcatctgt ctgtctgctg gtcatcgtgg
     1081 ggcttctcat gtttaggtac cataggagag gaggttatga tgtggaaaat gaagagaaag
     1141 tgaagttggg catgactaat tcccacatgg tgagcaaggg cgaggagctg ttcaccgggg
     1201 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     1261 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     1321 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     1381 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     1441 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     1501 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     1561 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     1621 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     1681 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     1741 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     1801 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     1861 ttggcatgga cgagctgtac aaaggcagtg gagagggcag aggcagtctg ctaacatgcg
     1921 gtgacgtcga ggagaatcct ggcccaatga ccgagtacaa gcccacggtg cgcctcgcca
     1981 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     2041 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     2101 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     2161 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     2221 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     2281 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc
     2341 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     2401 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccacaacctc cccttctacg
     2461 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     2521 gcatgacccg caagcccggt gcctgacgct gatcagcctc gactgtgcct tctagttgcc
     2581 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     2641 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     2701 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     2761 atgctgggga tgcggtgggc tctatggatg ccggatgttc ccgaagatgg gcggccgcca
     2821 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     2881 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     2941 ccttcccaac agttgcgcag cctgaatggc gaatggaaat tgtaagcgtt aatattttgt
     3001 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     3061 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     3121 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     3181 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     3241 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     3301 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     3361 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     3421 tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3481 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3541 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3601 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3661 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3721 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3781 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     3841 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3901 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3961 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     4021 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     4081 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     4141 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     4201 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4261 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     4321 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4381 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4441 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4501 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4561 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4621 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4681 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4741 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4801 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4861 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4921 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4981 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5041 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5101 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5161 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5221 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5281 gtattaccgc ctttgagtga gc
//
