LOCUS       sequence-382900-        5652 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bicistronic HSV-TK and mScarlet resistance for positive/negative 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5652)
  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 5652)
  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: pFB2F
FEATURES             Location/Qualifiers
     source          1..5652
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(3..26)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     376..395
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     401..420
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        401..419
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             423..1550
                     /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             1560..1613
                     /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             1614..2309
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(1747..1765)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2015..2034
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2328..2348
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2352..2372
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2376..2396
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2545..2562)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2551..2775
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2808..2825)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2808..2824)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2817..2839)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2965..3420)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3102..3123)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3314..3333
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3447..3551
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3552..3620,3621..4412)
                     /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(3770..3789)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4583..5171
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5072..5091
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5325..5342
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5459..5480
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5495..5500,5501..5518,5519..5525)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5533..5549
                     /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     5538..5560
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5557..5573
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5557..5573
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gagcatgcgc tttagcagcc ccgctgggca cttggcgcta cacaagtggc ctctggcctc
       61 gcacacattc cacatccacc ggtaggcgcc aaccggctcc gttctttggt ggccccttcg
      121 cgccaccttc tactcctccc ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt
      181 gcaggacgtg acaaatggaa gtagcacgtc tcactagtct cgtgcagatg gacagcaccg
      241 ctgagcaatg gaagcgggta ggcctttggg gcagcggcca atagcagctt tgctccttcg
      301 ctttctgggc tcagaggctg ggaaggggcg ggctcagggg cggggcgggc gcccgaaggt
      361 cctccggagg cccggcattc tgcacgcttc aaaagtctag taatacgact cactataggg
      421 ggatggcttc gtaccccggc catcagcacg cgtctgcgtt cgaccaggct gcgcgttctc
      481 gcggccatag caaccgacgt acggcgttgc gccctcgccg gcagcaagaa gccacggaag
      541 tccgcccgga gcagaaaatg cccacgctac tgcgggttta tatagacggt ccccacggga
      601 tggggaaaac caccaccacg caactgctgg tggccctggg ttcgcgcgac gatatcgtct
      661 acgtacccga gccgatgact tactggcggg tgctgggggc ttccgagaca atcgcgaaca
      721 tctacaccac acaacaccgc cttgaccagg gtgagatatc ggccggggac gcggcggtgg
      781 taatgacaag cgcccagata acaatgggca tgccttatgc cgtgaccgac gccgttctgg
      841 ctcctcatat cgggggggag gctgggagct cacatgcccc gcccccggcc ctcaccctca
      901 tcttcgaccg ccatcccatc gccgccctcc tgtgctaccc ggccgcgcga taccttatgg
      961 gcagcatgac cccccaggcc gtgctggcgt tcgtggccct catcccgccg accttgcccg
     1021 gcacaaacat cgtgttgggg gcccttccgg aggacagaca catcgaccgc ctggccaaac
     1081 gccagcgccc cggcgagcgg cttgacctgg ctatgctggc cgcgattcgc cgcgtttacg
     1141 ggctgcttgc caatacggtg cggtatctgc agggcggcgg gtcgtggcgg gaggattggg
     1201 gacagctttc ggggacggcc gtgccgcccc agggtgccga gccccagagc aacgcgggcc
     1261 cacgacccca tatcggggac acgttattta ccctgtttcg ggcccccgag ttgctggccc
     1321 ccaacggcga cctgtacaac gtgtttgcct gggccttgga cgtcttggcc aaacgcctcc
     1381 gtcccatgca cgtctttatc ctggattacg accaatcgcc cgccggctgc cgggacgccc
     1441 tgctgcaact tacctccggg atgatccaga cccacgtcac caccccaggc tccataccga
     1501 cgatctgcga cctggcgcgc acgtttgccc gggagatggg ggaggctaac ggcagtggag
     1561 agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc ccaatggtga
     1621 gcaagggcga ggcagtgatc aaggagttca tgcggttcaa ggtgcacatg gagggctcca
     1681 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     1741 agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttctcctgg gacatcctgt
     1801 cccctcagtt catgtacggc tccagggcct tcaccaagca ccccgccgac atccccgact
     1861 actataagca gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     1921 gcggcgccgt gaccgtgacc caggacacct ccctggagga cggcaccctg atctacaagg
     1981 tgaagctccg cggcaccaac ttccctcctg acggccccgt aatgcagaag aagacaatgg
     2041 gctgggaagc gtccaccgag cggttgtacc ccgaggacgg cgtgctgaag ggcgacatta
     2101 agatggccct gcgcctgaag gacggcggcc gctacctggc ggacttcaag accacctaca
     2161 aggccaagaa gcccgtgcag atgcccggcg cctacaacgt cgaccgcaag ttggacatca
     2221 cctcccacaa cgaggactac accgtggtgg aacagtacga acgctccgag ggccgccact
     2281 ccaccggcgg catggacgag ctgtacaagc tcagatctcg agctgatcca aaaaagaaga
     2341 gaaaggtaga tccgaagaaa aagcgtaaag ttgatcccaa gaaaaaacgg aaagtgggat
     2401 ccaccggatc tagccatggc ttcccgccgg aggtggagga gcaggatgat ggcacgctgc
     2461 ccatgtcttg tgcccaggag agcgggatgg accgtcaccc tgcagcctgt gcttctgcta
     2521 ggatcaatgt gtagcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     2581 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     2641 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     2701 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     2761 cggtgggctc tatggatgcc ggatgttccc gaagatgggc ggccgccact ggccgtcgtt
     2821 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     2881 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     2941 ttgcgcagcc tgaatggcga atggaaattg taagcgttaa tattttgtta aaattcgcgt
     3001 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     3061 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     3121 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     3181 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     3241 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     3301 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     3361 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     3421 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3481 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3541 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     3601 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     3661 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     3721 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     3781 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     3841 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     3901 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     3961 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     4021 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     4081 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     4141 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     4201 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     4261 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     4321 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     4381 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     4441 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     4501 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4561 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4621 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4681 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     4741 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4801 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4861 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4921 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4981 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5041 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5101 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5161 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5221 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     5281 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     5341 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     5401 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     5461 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     5521 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgagg
     5581 cgcgcccagg agctattaat tcgcggaggg aattctaccg ggtaggggag gcgcttttcc
     5641 caaggcagtc tg
//
