LOCUS       sequence-382892-        5806 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bicistronic HSV-TK-EGFP fusion and Puromycin resistance for 
            positive/negative selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5806)
  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 5806)
  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: pFB2E
FEATURES             Location/Qualifiers
     source          1..5806
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     71..88
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    205..226
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(241..246,247..264,265..271)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    279..295
                     /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     284..306
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     303..319
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     303..319
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        362..573
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      573..582
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             579..1706
                     /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             join(1707..1709,1710..1712,1713..2423)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1707..1709,1710..1712,1713..2423)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1752..1773)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2013..2032)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2433..2486
                     /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             2487..3086
                     /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(2487..2506)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(3097..3114)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3103..3327
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3360..3377)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3360..3376)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3369..3391)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(3517..3972)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3654..3675)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3866..3885
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3999..4103
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4104..4172,4173..4964)
                     /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(4322..4341)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5135..5723
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5624..5643
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
       61 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
      121 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
      181 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
      241 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
      301 cacaggaaac agctatgacc atgaggcgcg cccaggagct attaattcgc ggagggaatt
      361 cgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
      421 accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
      481 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
      541 ctttttcgca acgggtttgc cgccagaaca cagccaccat ggcttcgtac cccggccatc
      601 agcacgcgtc tgcgttcgac caggctgcgc gttctcgcgg ccatagcaac cgacgtacgg
      661 cgttgcgccc tcgccggcag caagaagcca cggaagtccg cccggagcag aaaatgccca
      721 cgctactgcg ggtttatata gacggtcccc acgggatggg gaaaaccacc accacgcaac
      781 tgctggtggc cctgggttcg cgcgacgata tcgtctacgt acccgagccg atgacttact
      841 ggcgggtgct gggggcttcc gagacaatcg cgaacatcta caccacacaa caccgccttg
      901 accagggtga gatatcggcc ggggacgcgg cggtggtaat gacaagcgcc cagataacaa
      961 tgggcatgcc ttatgccgtg accgacgccg ttctggctcc tcatatcggg ggggaggctg
     1021 ggagctcaca tgccccgccc ccggccctca ccctcatctt cgaccgccat cccatcgccg
     1081 ccctcctgtg ctacccggcc gcgcgatacc ttatgggcag catgaccccc caggccgtgc
     1141 tggcgttcgt ggccctcatc ccgccgacct tgcccggcac aaacatcgtg ttgggggccc
     1201 ttccggagga cagacacatc gaccgcctgg ccaaacgcca gcgccccggc gagcggcttg
     1261 acctggctat gctggccgcg attcgccgcg tttacgggct gcttgccaat acggtgcggt
     1321 atctgcaggg cggcgggtcg tggcgggagg attggggaca gctttcgggg acggccgtgc
     1381 cgccccaggg tgccgagccc cagagcaacg cgggcccacg accccatatc ggggacacgt
     1441 tatttaccct gtttcgggcc cccgagttgc tggcccccaa cggcgacctg tacaacgtgt
     1501 ttgcctgggc cttggacgtc ttggccaaac gcctccgtcc catgcacgtc tttatcctgg
     1561 attacgacca atcgcccgcc ggctgccggg acgccctgct gcaacttacc tccgggatga
     1621 tccagaccca cgtcaccacc ccaggctcca taccgacgat ctgcgacctg gcgcgcacgt
     1681 ttgcccggga gatgggggag gctaacatgg tgagcaaggg cgaggagctg ttcaccgggg
     1741 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     1801 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     1861 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     1921 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     1981 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     2041 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2101 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     2161 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     2221 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     2281 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     2341 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     2401 ttggcatgga cgagctgtac aaaggcagtg gagagggcag aggaagtctg ctaacatgcg
     2461 gtgacgtcga ggagaatcct ggcccaatga ccgagtacaa gcccacggtg cgcctcgcca
     2521 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     2581 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     2641 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     2701 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     2761 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     2821 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc
     2881 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     2941 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccacaacctc cccttctacg
     3001 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     3061 gcatgacccg caagcccggt gcctgacgct gatcagcctc gactgtgcct tctagttgcc
     3121 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     3181 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     3241 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     3301 atgctgggga tgcggtgggc tctatggatg ccggatgttc ccgaagatgg gcggccgcca
     3361 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     3421 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     3481 ccttcccaac agttgcgcag cctgaatggc gaatggaaat tgtaagcgtt aatattttgt
     3541 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     3601 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     3661 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     3721 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     3781 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     3841 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     3901 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     3961 tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     4021 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     4081 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     4141 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     4201 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     4261 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     4321 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     4381 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     4441 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     4501 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     4561 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     4621 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     4681 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     4741 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4801 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     4861 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4921 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4981 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     5041 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     5101 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5161 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5221 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5281 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5341 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5401 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5461 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5521 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5581 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5641 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5701 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5761 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctga
//
