LOCUS       sequence-382884-        5331 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bicistronic HSV-TK and Puromycin resistance for positive/negative 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5331)
  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 5331)
  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: pFB2B
FEATURES             Location/Qualifiers
     source          1..5331
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     372..391
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     397..416
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        397..415
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             419..1546
                     /codon_start=1
                     /product="herpes simplex virus thymidine kinase"
                     /label=HSV TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MASYPGHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRPEQK
                     MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRVLGASETIANIYTTQ
                     HRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDR
                     HPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQR
                     PGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAVPPQGAEPQSNAGPR
                     PHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDAL
                     LQLTSGMIQTHVTTPGSIPTICDLARTFAREMGEAN"
     CDS             1556..1609
                     /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             1610..2209
                     /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(1610..1629)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(2220..2237)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2226..2450
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2483..2500)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2483..2499)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2492..2514)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2640..3095)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2777..2798)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2989..3008
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3122..3226
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3227..3295,3296..4087)
                     /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(3445..3464)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4258..4846
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4747..4766
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5000..5017
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5134..5155
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5170..5175,5176..5193,5194..5200)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5208..5224
                     /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     5213..5235
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5232..5248
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5232..5248
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(join(5330..5331,1..22))
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
ORIGIN
        1 atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac
       61 acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc
      121 accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag
      181 gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga
      241 gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt
      301 ctgggctcag aggctgggaa ggggcgggct caggggcggg gcgggcgccc gaaggtcctc
      361 cggaggcccg gcattctgca cgcttcaaaa gtctagtaat acgactcact atagggggat
      421 ggcttcgtac cccggccatc agcacgcgtc tgcgttcgac caggctgcgc gttctcgcgg
      481 ccatagcaac cgacgtacgg cgttgcgccc tcgccggcag caagaagcca cggaagtccg
      541 cccggagcag aaaatgccca cgctactgcg ggtttatata gacggtcccc acgggatggg
      601 gaaaaccacc accacgcaac tgctggtggc cctgggttcg cgcgacgata tcgtctacgt
      661 acccgagccg atgacttact ggcgggtgct gggggcttcc gagacaatcg cgaacatcta
      721 caccacacaa caccgccttg accagggtga gatatcggcc ggggacgcgg cggtggtaat
      781 gacaagcgcc cagataacaa tgggcatgcc ttatgccgtg accgacgccg ttctggctcc
      841 tcatatcggg ggggaggctg ggagctcaca tgccccgccc ccggccctca ccctcatctt
      901 cgaccgccat cccatcgccg ccctcctgtg ctacccggcc gcgcgatacc ttatgggcag
      961 catgaccccc caggccgtgc tggcgttcgt ggccctcatc ccgccgacct tgcccggcac
     1021 aaacatcgtg ttgggggccc ttccggagga cagacacatc gaccgcctgg ccaaacgcca
     1081 gcgccccggc gagcggcttg acctggctat gctggccgcg attcgccgcg tttacgggct
     1141 gcttgccaat acggtgcggt atctgcaggg cggcgggtcg tggcgggagg attggggaca
     1201 gctttcgggg acggccgtgc cgccccaggg tgccgagccc cagagcaacg cgggcccacg
     1261 accccatatc ggggacacgt tatttaccct gtttcgggcc cccgagttgc tggcccccaa
     1321 cggcgacctg tacaacgtgt ttgcctgggc cttggacgtc ttggccaaac gcctccgtcc
     1381 catgcacgtc tttatcctgg attacgacca atcgcccgcc ggctgccggg acgccctgct
     1441 gcaacttacc tccgggatga tccagaccca cgtcaccacc ccaggctcca taccgacgat
     1501 ctgcgacctg gcgcgcacgt ttgcccggga gatgggggag gctaacggca gtggagaggg
     1561 cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccaa tgaccgagta
     1621 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc
     1681 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga
     1741 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
     1801 gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc
     1861 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
     1921 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
     1981 cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
     2041 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc
     2101 gccccacaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
     2161 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgac gctgatcagc
     2221 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     2281 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2341 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2401 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg atgccggatg
     2461 ttcccgaaga tgggcggccg ccactggccg tcgttttaca acgtcgtgac tgggaaaacc
     2521 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     2581 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
     2641 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     2701 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     2761 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     2821 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     2881 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     2941 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     3001 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     3061 acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt
     3121 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     3181 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     3241 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     3301 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     3361 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     3421 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     3481 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     3541 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     3601 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     3661 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     3721 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     3781 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     3841 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     3901 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     3961 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     4021 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     4081 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     4141 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     4201 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     4261 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     4321 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4381 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     4441 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4501 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4561 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4621 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     4681 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     4741 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     4801 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     4861 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     4921 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     4981 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     5041 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     5101 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
     5161 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
     5221 acaatttcac acaggaaaca gctatgacca tgaggcgcgc ccaggagcta ttaattcgcg
     5281 gagggaattc taccgggtag gggaggcgct tttcccaagg cagtctggag c
//
