LOCUS       sequence-382902-        5655 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bicistronic HSV-TK and mTagBFP resistance for positive/negative 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5655)
  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 5655)
  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: pFB2G
FEATURES             Location/Qualifiers
     source          1..5655
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(25..48)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     398..417
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     423..442
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        423..441
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             445..1572
                     /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             1582..1635
                     /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             1636..2334
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             2353..2373
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2377..2397
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2401..2421
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2570..2587)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2576..2800
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2833..2850)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2833..2849)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2842..2864)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2990..3445)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3127..3148)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3339..3358
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3472..3576
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3577..3645,3646..4437)
                     /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(3795..3814)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4608..5196
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5097..5116
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5350..5367
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5484..5505
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5520..5525,5526..5543,5544..5550)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5558..5574
                     /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     5563..5585
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5582..5598
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5582..5598
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg cacttggcgc
       61 tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct
      121 ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc
      181 cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcactagt
      241 ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc
      301 caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg cgggctcagg
      361 ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat tctgcacgct tcaaaagtct
      421 agtaatacga ctcactatag ggggatggct tcgtaccccg gccatcagca cgcgtctgcg
      481 ttcgaccagg ctgcgcgttc tcgcggccat agcaaccgac gtacggcgtt gcgccctcgc
      541 cggcagcaag aagccacgga agtccgcccg gagcagaaaa tgcccacgct actgcgggtt
      601 tatatagacg gtccccacgg gatggggaaa accaccacca cgcaactgct ggtggccctg
      661 ggttcgcgcg acgatatcgt ctacgtaccc gagccgatga cttactggcg ggtgctgggg
      721 gcttccgaga caatcgcgaa catctacacc acacaacacc gccttgacca gggtgagata
      781 tcggccgggg acgcggcggt ggtaatgaca agcgcccaga taacaatggg catgccttat
      841 gccgtgaccg acgccgttct ggctcctcat atcggggggg aggctgggag ctcacatgcc
      901 ccgcccccgg ccctcaccct catcttcgac cgccatccca tcgccgccct cctgtgctac
      961 ccggccgcgc gataccttat gggcagcatg accccccagg ccgtgctggc gttcgtggcc
     1021 ctcatcccgc cgaccttgcc cggcacaaac atcgtgttgg gggcccttcc ggaggacaga
     1081 cacatcgacc gcctggccaa acgccagcgc cccggcgagc ggcttgacct ggctatgctg
     1141 gccgcgattc gccgcgttta cgggctgctt gccaatacgg tgcggtatct gcagggcggc
     1201 gggtcgtggc gggaggattg gggacagctt tcggggacgg ccgtgccgcc ccagggtgcc
     1261 gagccccaga gcaacgcggg cccacgaccc catatcgggg acacgttatt taccctgttt
     1321 cgggcccccg agttgctggc ccccaacggc gacctgtaca acgtgtttgc ctgggccttg
     1381 gacgtcttgg ccaaacgcct ccgtcccatg cacgtcttta tcctggatta cgaccaatcg
     1441 cccgccggct gccgggacgc cctgctgcaa cttacctccg ggatgatcca gacccacgtc
     1501 accaccccag gctccatacc gacgatctgc gacctggcgc gcacgtttgc ccgggagatg
     1561 ggggaggcta acggcagtgg agagggcaga ggaagtctgc taacatgcgg tgacgttgag
     1621 gagaatcctg gcccaatgag cgagctgatt aaggagaaca tgcacatgaa gctgtacatg
     1681 gagggcaccg tggacaacca tcacttcaag tgcacatccg agggcgaagg caagccctac
     1741 gagggcaccc agaccatgag aatcaaggtg gtcgagggcg gccctctccc cttcgccttc
     1801 gacatcctgg ctactagctt cctctacggc agcaagacct tcatcaacca cacccagggc
     1861 atccccgact tcttcaagca gtccttccct gagggcttca catgggagag agtcaccaca
     1921 tacgaagacg ggggcgtgct gaccgctacc caggacacca gcctccagga cggctgcctc
     1981 atctacaacg tcaagatcag aggggtgaac ttcacatcca acggccctgt gatgcagaag
     2041 aaaacactcg gctgggaggc cttcaccgaa acgctgtacc ccgctgacgg cggcctggaa
     2101 ggcagaaacg acatggccct gaagctcgtg ggcgggagcc atctgatcgc aaacatcaag
     2161 accacatata gatccaagaa acccgctaag aacctcaaga tgcctggcgt ctactatgtg
     2221 gactacagac tggaaagaat caaggaggcc aacaacgaga cctacgtcga gcagcacgag
     2281 gtggcagtgg ccagatactg cgacctccct agcaaactgg ggcacaagct taatctcaga
     2341 tctcgagctg atccaaaaaa gaagagaaag gtagatccga agaaaaagcg taaagttgat
     2401 cccaagaaaa aacggaaagt gggatccact ggatctagcc atggcttccc gccggaggtg
     2461 gaggagcagg atgatggcac gctgcccatg tcttgtgccc aggagagcgg gatggaccgt
     2521 caccctgcag cctgtgcttc tgctaggatc aatgtgtagc gctgatcagc ctcgactgtg
     2581 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     2641 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     2701 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     2761 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg atgccggatg ttcccgaaga
     2821 tgggcggccg ccactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     2881 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     2941 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga aattgtaagc
     3001 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     3061 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     3121 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     3181 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     3241 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     3301 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     3361 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     3421 cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     3481 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3541 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     3601 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     3661 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     3721 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     3781 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     3841 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     3901 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     3961 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     4021 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     4081 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     4141 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     4201 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     4261 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     4321 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     4381 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     4441 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     4501 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     4561 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4621 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4681 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4741 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4801 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4861 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4921 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4981 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5041 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5101 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5161 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5221 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5281 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     5341 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     5401 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     5461 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     5521 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     5581 acaggaaaca gctatgacca tgaggcgcgc ccaggagcta ttaattcgcg gagggaattc
     5641 taccgggtag gggag
//
