LOCUS       sequence-382879-        5783 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bicistronic HSV-TK and Hygromycin resistance for positive/negative 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5783)
  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 5783)
  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: pFB2C
FEATURES             Location/Qualifiers
     source          1..5783
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     88..105
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    222..243
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(258..263,264..281,282..288)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    296..312
                     /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     301..323
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     320..336
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     320..336
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(418..441)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     791..810
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     816..835
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        816..834
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             838..1965
                     /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             1975..2028
                     /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             2029..3054
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2029..3054
                     /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    3097..3321
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3354..3371)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3354..3370)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3363..3385)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(3511..3966)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3648..3669)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3860..3879
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3993..4097
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4098..4166,4167..4958)
                     /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(4316..4335)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5129..5717
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5618..5637
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
       61 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
      121 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
      181 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
      241 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
      301 agcggataac aatttcacac aggaaacagc tatgaccatg aggcgcgccc aggagctatt
      361 aattcgcgga gggaattcta ccgggtaggg gaggcgcttt tcccaaggca gtctggagca
      421 tgcgctttag cagccccgct gggcacttgg cgctacacaa gtggcctctg gcctcgcaca
      481 cattccacat ccaccggtag gcgccaaccg gctccgttct ttggtggccc cttcgcgcca
      541 ccttctactc ctcccctagt caggaagttc ccccccgccc cgcagctcgc gtcgtgcagg
      601 acgtgacaaa tggaagtagc acgtctcact agtctcgtgc agatggacag caccgctgag
      661 caatggaagc gggtaggcct ttggggcagc ggccaatagc agctttgctc cttcgctttc
      721 tgggctcaga ggctgggaag gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc
      781 ggaggcccgg cattctgcac gcttcaaaag tctagtaata cgactcacta tagggggatg
      841 gcttcgtacc ccggccatca gcacgcgtct gcgttcgacc aggctgcgcg ttctcgcggc
      901 catagcaacc gacgtacggc gttgcgccct cgccggcagc aagaagccac ggaagtccgc
      961 ccggagcaga aaatgcccac gctactgcgg gtttatatag acggtcccca cgggatgggg
     1021 aaaaccacca ccacgcaact gctggtggcc ctgggttcgc gcgacgatat cgtctacgta
     1081 cccgagccga tgacttactg gcgggtgctg ggggcttccg agacaatcgc gaacatctac
     1141 accacacaac accgccttga ccagggtgag atatcggccg gggacgcggc ggtggtaatg
     1201 acaagcgccc agataacaat gggcatgcct tatgccgtga ccgacgccgt tctggctcct
     1261 catatcgggg gggaggctgg gagctcacat gccccgcccc cggccctcac cctcatcttc
     1321 gaccgccatc ccatcgccgc cctcctgtgc tacccggccg cgcgatacct tatgggcagc
     1381 atgacccccc aggccgtgct ggcgttcgtg gccctcatcc cgccgacctt gcccggcaca
     1441 aacatcgtgt tgggggccct tccggaggac agacacatcg accgcctggc caaacgccag
     1501 cgccccggcg agcggcttga cctggctatg ctggccgcga ttcgccgcgt ttacgggctg
     1561 cttgccaata cggtgcggta tctgcagggc ggcgggtcgt ggcgggagga ttggggacag
     1621 ctttcgggga cggccgtgcc gccccagggt gccgagcccc agagcaacgc gggcccacga
     1681 ccccatatcg gggacacgtt atttaccctg tttcgggccc ccgagttgct ggcccccaac
     1741 ggcgacctgt acaacgtgtt tgcctgggcc ttggacgtct tggccaaacg cctccgtccc
     1801 atgcacgtct ttatcctgga ttacgaccaa tcgcccgccg gctgccggga cgccctgctg
     1861 caacttacct ccgggatgat ccagacccac gtcaccaccc caggctccat accgacgatc
     1921 tgcgacctgg cgcgcacgtt tgcccgggag atgggggagg ctaacggcag tggagagggc
     1981 agaggaagtc tgctaacatg cggtgacgtc gaggagaatc ctggcccaat gaaaaagcct
     2041 gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac
     2101 ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt
     2161 ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat
     2221 cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaatttagc
     2281 gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct
     2341 gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg
     2401 gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac
     2461 actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact
     2521 gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg
     2581 gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc
     2641 ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat
     2701 tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag
     2761 cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg
     2821 tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat
     2881 gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc
     2941 gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta
     3001 ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga ataggcacag
     3061 tcgaggctga tcagcgagct cgctgatcag cctcgactgt gccttctagt tgccagccat
     3121 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     3181 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     3241 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     3301 gggatgcggt gggctctatg gatgccggat gttcccgaag atgggcggcc gccactggcc
     3361 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     3421 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     3481 caacagttgc gcagcctgaa tggcgaatgg aaattgtaag cgttaatatt ttgttaaaat
     3541 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     3601 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     3661 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     3721 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     3781 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     3841 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     3901 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     3961 gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     4021 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     4081 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     4141 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     4201 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     4261 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     4321 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     4381 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     4441 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     4501 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     4561 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     4621 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     4681 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     4741 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     4801 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     4861 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     4921 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     4981 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5041 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5101 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     5161 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     5221 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     5281 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     5341 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5401 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5461 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5521 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5581 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5641 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5701 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5761 ggccttttgc tcacatgttc ttt
//
