LOCUS       sequence-382881-        5279 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bicistronic DTR and Hygromycin resistance for positive/negative 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5279)
  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 5279)
  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: pFB2A
FEATURES             Location/Qualifiers
     source          1..5279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     95..112
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    229..250
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(265..270,271..288,289..295)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    303..319
                     /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     308..330
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     327..343
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     327..343
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(425..448)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     798..817
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     823..842
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        823..841
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1478..1531
                     /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             1532..2557
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1532..2557
                     /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"
     polyA_signal    2600..2824
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2857..2874)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2857..2873)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2866..2888)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(3014..3469)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3151..3172)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3363..3382
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3496..3600
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3601..3669,3670..4461)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGQDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3819..3838)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4632..5220
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5121..5140
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
       61 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
      121 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
      181 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
      241 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
      301 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgagg cgcgcccagg
      361 agctattaat tcgcggaggg aattctaccg ggtaggggag gcgcttttcc caaggcagtc
      421 tggagcatgc gctttagcag ccccgctggg cacttggcgc tacacaagtg gcctctggcc
      481 tcgcacacat tccacatcca ccggtaggcg ccaaccggct ccgttctttg gtggcccctt
      541 cgcgccacct tctactcctc ccctagtcag gaagttcccc cccgccccgc agctcgcgtc
      601 gtgcaggacg tgacaaatgg aagtagcacg tctcactagt ctcgtgcaga tggacagcac
      661 cgctgagcaa tggaagcggg taggcctttg gggcagcggc caatagcagc tttgctcctt
      721 cgctttctgg gctcagaggc tgggaagggg cgggctcagg ggcggggcgg gcgcccgaag
      781 gtcctccgga ggcccggcat tctgcacgct tcaaaagtct agtaatacga ctcactatag
      841 ggggatgaag ctgctgccgt cggtggtgct gaagctcttt ctggctgcag ttctctcggc
      901 actggtgact ggcgagagcc tggagcggct tcggagaggg ctagctgctg gaaccagcaa
      961 cccggaccct cccactgtat ccacggacca gctgctaccc ctaggaggcg gccgggaccg
     1021 gaaagtccgt gacttgcaag aggcagatct ggaccttttg agagtcactt tatcctccaa
     1081 gccacaagca ctggccacac caaacaagga ggagcacggg aaaagaaaga agaaaggcaa
     1141 ggggctaggg aagaagaggg acccatgtct tcggaaatac aaggacttct gcatccatgg
     1201 agaatgcaaa tatgtgaagg agctccgggc tccctcctgc atctgccacc cgggttacca
     1261 tggagagagg tgtcatgggc tgagcctccc agtggaaaat cgcttatata cctatgacca
     1321 cacaaccatc ctggccgtgg tggctgtggt gctgtcatct gtctgtctgc tggtcatcgt
     1381 ggggcttctc atgtttaggt accataggag aggaggttat gatgtggaaa atgaagagaa
     1441 agtgaagttg ggcatgacta attcccacgg cagtggagag ggcagaggaa gtctgctaac
     1501 atgcggtgac gtcgaggaga atcctggccc aatgaaaaag cctgaactca ccgcgacgtc
     1561 tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga
     1621 gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt
     1681 aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc
     1741 cgcgctcccg attccggaag tgcttgacat tggggaattt agcgagagcc tgacctattg
     1801 catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc
     1861 tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac
     1921 gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt
     1981 catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt
     2041 cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga
     2101 agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg
     2161 cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc
     2221 caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga
     2281 gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg
     2341 tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca
     2401 gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc
     2461 ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa
     2521 ccgacgcccc agcactcgtc cgagggcaaa ggaataggca cagtcgaggc tgatcagcga
     2581 gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     2641 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     2701 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     2761 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     2821 atggatgccg gatgttcccg aagatgggcg gccgccactg gccgtcgttt tacaacgtcg
     2881 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     2941 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     3001 gaatggcgaa tggaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     3061 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     3121 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     3181 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     3241 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     3301 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     3361 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     3421 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact
     3481 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3541 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3601 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3661 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     3721 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     3781 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     3841 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     3901 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     3961 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     4021 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     4081 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     4141 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     4201 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     4261 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     4321 cgcggtatca ttgcagcact ggggcaagat ggtaagccct cccgtatcgt agttatctac
     4381 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     4441 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     4501 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4561 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4621 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4681 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4741 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     4801 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4861 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4921 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4981 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     5041 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     5101 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     5161 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     5221 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacat
//
