LOCUS       sequence-228301-        6699 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6699)
  AUTHORS   Boehm JS, Hession MT, Bulmer SE, Hahn WC
  TITLE     Transformation of human and murine fibroblasts without viral 
            oncoproteins.
  JOURNAL   Mol Cell Biol. 2005 Aug . 25(15):6464-74.
  PUBMED    16024784
REFERENCE   2  (bases 1 to 6699)
  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"; journalName: "Mol Cell 
            Biol. 2005 Aug . 25(15):6464-74."
COMMENT     Sequence Label: pMKO.1 puro Rb shRNA
FEATURES             Location/Qualifiers
     source          1..6699
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     116..134
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        234..613
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        614..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     768..788
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             818..993
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    1056..1255
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1456..1872
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1802..1824
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1840..1856
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1908..2156
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1908..1928
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2079..2098
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        2246..2575
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2426..2561
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2585..3184
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2585..2604)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3081..3101
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3380..3805
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(3908..3930)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4071..4400
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4251..4386
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4422..4441
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4813..5401)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4893..4912)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5572..6363,6364..6432))
                     /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     6195..6214
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6433..6537)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
       61 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
      121 gcgtatcacg aggccctttc gtcttcaaga attagcttgg ccattgcata cgttgtatcc
      181 atatcataat atgtacattt atattggctc atgtccaaca ttaccgccat gttgacattg
      241 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
      301 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
      361 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
      421 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      481 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
      541 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      601 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
      661 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
      721 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
      781 taggcgtgta cggtgggagg tctatataag cagagctcaa taaaagagcc cacaacccct
      841 cactcggcgc gccagtcttc cgatagactg cgtcgcccgg gtacccgtat tcccaataaa
      901 gcctcttgct gtttgcatcc gaatcgtggt ctcgctgttc cttgggaggg tctcctctga
      961 gtgattgact acccacgacg ggggtctttc atttgggggc tcgtccggga tttggagacc
     1021 cctgcccagg gaccaccgac ccaccaccgg gaggtaagct ggccagcaac ttatctgtgt
     1081 ctgtccgatt gtctagtgtc tatgtttgat gttatgcgcc tgcgtctgta ctagttagct
     1141 aactagctct gtatctggcg gacccgtggt ggaactgacg agttctgaac acccggccgc
     1201 aaccctggga gacgtcccag ggactttggg ggccgttttt gtggcccgac ctgaggaagg
     1261 gagtcgatgt ggaatccgac cccgtcagga tatgtggttc tggtaggaga cgagaaccta
     1321 aaacagttcc cgcctccgtc tgaatttttg ctttcggttt ggaaccgaag ccgcgcgtct
     1381 tgtctgctgc agcgctgcag catcgttctg tgttgtctct gtctgactgt gtttctgtat
     1441 ttgtctgaaa attagggcca gactgttacc actcccttaa gtttgacctt aggtcactgg
     1501 aaagatgtcg agcggatcgc tcacaaccag tcggtagatg tcaagaagag acgttgggtt
     1561 accttctgct ctgcagaatg gccaaccttt aacgtcggat ggccgcgaga cggcaccttt
     1621 aaccgagacc tcatcaccca ggttaagatc aaggtctttt cacctggccc gcatggacac
     1681 ccagaccagg tcccctacat cgtgacctgg gaagccttgg cttttgaccc ccctccctgg
     1741 gtcaagccct ttgtacaccc taagcctccg cctcctcttc ctccatccgc cccgtctctc
     1801 ccccttgaac ctcctcgttc gaccccgcct cgatcctccc tttatccagc cctcactcct
     1861 tctctaggcg ccggaattga agatctgtcg acaaggtcgg gcaggaagag ggcctatttc
     1921 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attagaatta
     1981 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     2041 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     2101 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccggca
     2161 gagatcgtgt attgagattc tcgagaatct caatacacga tctctgtttt tgaattcgcc
     2221 agcacagtgg tcgaccctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctt
     2281 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     2341 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     2401 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     2461 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     2521 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     2581 taccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc
     2641 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc
     2701 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct
     2761 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc
     2821 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag
     2881 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa
     2941 ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct
     3001 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
     3061 cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac
     3121 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
     3181 ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac cccatgcatc
     3241 gattagtcca atttgttaaa gacaggatat cagtggtcca ggctctagtt ttgactcaac
     3301 aatatcacca gctgaagcct atagagtacg agccatagat aaaataaaag attttattta
     3361 gtctccagaa aaagggggga atgaaagacc ccacctgtag gtttggcaag ctagcttaag
     3421 taacgccatt ttgcaaggca tggaaaaata cataactgag aatagagaag ttcagatcaa
     3481 ggtcaggaac agatggaaca gggtcgaccc tagagaacca tcagatgttt ccagggtgcc
     3541 ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc gcttctcgct
     3601 tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc ctcactcggg
     3661 gcgccagtcc tccgattgac tgagtcgccc gggtacccgt gtatccaata aaccctcttg
     3721 cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg gtctcctctg agtgattgac
     3781 tacccgtcag cgggggtctt tcatttgggg gctcgtccgg gatcgggaga cccctgccca
     3841 gggaccaccg acccaccacc gggaggtaag ctggctgcct cgcgcgtttc ggtgatgacg
     3901 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     3961 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag
     4021 ccatgaccca gtcacgtagc gatagcggag tgtagatccg gctgtggaat gtgtgtcagt
     4081 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     4141 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     4201 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     4261 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4321 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4381 gaggcctagg cttttgcaaa aagcttactg gcttaactat gcggcatcag agcagattgt
     4441 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     4501 catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4561 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4621 cgcaggaaag aacatggctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4681 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4741 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4801 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4861 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4921 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4981 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     5041 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5101 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     5161 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     5221 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     5281 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5341 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5401 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5461 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5521 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5581 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5641 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5701 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5761 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5821 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5881 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5941 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     6001 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     6061 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     6121 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     6181 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     6241 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     6301 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     6361 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6421 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6481 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6541 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta
     6601 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttaa
     6661 ttattgaagc atttatcagg gttattgtct catgagcgg
//
