LOCUS       sequence-382895-        4882 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bicistronic DTR and Puromycin resistance for positive/negative 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4882)
  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 4882)
  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: pFB2
FEATURES             Location/Qualifiers
     source          1..4882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        23..127
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(128..196,197..988)
                     /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(346..365)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1159..1747
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1648..1667
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1901..1918
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2035..2056
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2071..2076,2077..2094,2095..2101)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2109..2125
                     /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     2114..2136
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2133..2149
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2133..2149
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2231..2254)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2604..2623
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     2684..2703
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2684..2702
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3339..3392
                     /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             3393..3992
                     /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(3393..3412)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(4003..4020)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4009..4233
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4266..4283)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4266..4282)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4275..4297)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(4423..4878)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4560..4581)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4772..4791
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
       61 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
      121 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
      181 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
      241 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
      301 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
      361 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
      421 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
      481 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
      541 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
      601 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
      661 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
      721 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
      781 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
      841 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
      901 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
      961 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     1021 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     1081 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     1141 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     1201 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     1261 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     1321 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     1381 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     1441 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     1501 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     1561 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     1621 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     1681 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     1741 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     1801 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     1861 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     1921 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     1981 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     2041 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
     2101 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgaggcgcg
     2161 cccaggagct attaattcgc ggagggaatt ctaccgggta ggggaggcgc ttttcccaag
     2221 gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca caagtggcct
     2281 ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt tctttggtgg
     2341 ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg ccccgcagct
     2401 cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg tgcagatgga
     2461 cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat agcagctttg
     2521 ctccttcgct ttctgggctc agaggctggg aaggggcggg ctcaggggcg gggcgggcgc
     2581 ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagtctagag cctctgctaa
     2641 ccatgttcat gccttcttct ttttcctaca gatccttaat taataatacg actcactata
     2701 gggggatgaa gctgctgccg tcggtggtgc tgaagctctt tctggctgca gttctctcgg
     2761 cactggtgac tggcgagagc ctggagcggc ttcggagagg gctagctgct ggaaccagca
     2821 acccggaccc tcccactgta tccacggacc agctgctacc cctaggaggc ggccgggacc
     2881 ggaaagtccg tgacttgcaa gaggcagatc tggacctttt gagagtcact ttatcctcca
     2941 agccacaagc actggccaca ccaaacaagg aggagcacgg gaaaagaaag aagaaaggca
     3001 aggggctagg gaagaagagg gacccatgtc ttcggaaata caaggacttc tgcatccatg
     3061 gagaatgcaa atatgtgaag gagctccggg ctccctcctg catctgccac ccgggttacc
     3121 atggagagag gtgtcatggg ctgagcctcc cagtggaaaa tcgcttatat acctatgacc
     3181 acacaaccat cctggccgtg gtggctgtgg tgctgtcatc tgtctgtctg ctggtcatcg
     3241 tggggcttct catgtttagg taccatagga gaggaggtta tgatgtggaa aatgaagaga
     3301 aagtgaagtt gggcatgact aattcccacg gcagtggaga gggcagagga agtctgctaa
     3361 catgcggtga cgtcgaggag aatcctggcc caatgaccga gtacaagccc acggtgcgcc
     3421 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact
     3481 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc
     3541 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     3601 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     3661 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     3721 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     3781 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     3841 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccac aacctcccct
     3901 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     3961 cctggtgcat gacccgcaag cccggtgcct gacgctgatc agcctcgact gtgccttcta
     4021 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     4081 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     4141 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     4201 gcaggcatgc tggggatgcg gtgggctcta tggatgccgg atgttcccga agatgggcgg
     4261 ccgccactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     4321 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     4381 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggaaattgta agcgttaata
     4441 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     4501 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     4561 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     4621 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     4681 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     4741 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     4801 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     4861 cgccgctaca gggcgcgtca gg
//
