LOCUS       sequence-142557-        4896 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 4896)
  AUTHORS   Tucker KL, Beard C, Dausmann J, Jackson-Grusby L, Laird PW, Lei H, 
            Li E, Jaenisch R
  TITLE     Germ-line passage is required for establishment of methylation and 
            expression patterns of imprinted but not of nonimprinted genes.
  JOURNAL   Genes Dev. 1996 Apr 15. 10(8):1008-20.
  PUBMED    8608936
REFERENCE   2  (bases 1 to 4896)
  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: "Genes Dev. 
            1996 Apr 15. 10(8):1008-20."
COMMENT     Sequence Label: pPGKpuro
FEATURES             Location/Qualifiers
     source          1..4896
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        55..554
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(90..113)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     487..506
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             594..1193
                     /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(594..613)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1090..1110
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     promoter        complement(1767..1785)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1768..1785)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1803..1819)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1803..1819)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1816..1838)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1827..1843
                     /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)."
     promoter        complement(join(1851..1857,1858..1875,1876..1881))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1896..1917
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2034..2051)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2205..2793)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2285..2304)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2964..3755,3756..3824))
                     /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     3587..3606
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3825..3929)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3997..4015
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4053..4075)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4175..4194
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4261..4716)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4398..4419)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4610..4629
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4842..4864
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4856..4873
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4857..4873
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(4880..4896,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4880..4896,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg gccaagtcgg ccgagctcga attcgtcgac ctcgaaattc taccgggtag
       61 gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg ctgggcactt
      121 ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt aggcgccaac
      181 cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta gtcaggaagt
      241 tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca
      301 ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc ctttggggca
      361 gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga aggggtgggt
      421 ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg tcctccggag
      481 gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc tcttcctcat
      541 ctccgggcct ttcgacctgc atccatctag atctcgagca gctgaagctt accatgaccg
      601 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc
      661 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca
      721 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
      781 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
      841 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
      901 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
      961 ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     1021 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct
     1081 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     1141 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc
     1201 cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatgcatcg atgatatcag
     1261 atccccggga tgcagaaatt gatgatctat taaacaataa agatgtccac taaaatggaa
     1321 gtttttcctg tcatactttg ttaagaaggg tgagaacaga gtacctacat tttgaatgga
     1381 aggattggag ctacgggggt gggggtgggg tgggattaga taaatgcctg ctctttactg
     1441 aaggctcttt actattgctt tatgataatg tttcatagtt ggatatcata atttaaacaa
     1501 gcaaaaccaa attaagggcc agctcattcc tcccactcat gatctataga tctatagatc
     1561 tctcgtggga tcattgtttt tctcttgatt cccactttgt ggttctaagt actgtggttt
     1621 ccaaatgtgt cagtttcata gcctgaagaa cgagatcagc agcctctgtt ccacatacac
     1681 ttcattctca gtattgtttt gccaagttct aattccatca gaagctggtc gactctagat
     1741 gcggccgcat gcataagctt gagtattcta tagtgtcacc taaatagctt ggcgtaatca
     1801 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     1861 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     1921 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     1981 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     2041 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     2101 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     2161 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     2221 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     2281 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     2341 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     2401 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2461 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     2521 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     2581 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     2641 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     2701 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     2761 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     2821 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     2881 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     2941 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     3001 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     3061 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     3121 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     3181 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     3241 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     3301 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     3361 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     3421 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3481 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     3541 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     3601 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     3661 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     3721 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     3781 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     3841 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     3901 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     3961 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     4021 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     4081 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     4141 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     4201 gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     4261 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     4321 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     4381 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     4441 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     4501 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     4561 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     4621 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     4681 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa
     4741 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     4801 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
     4861 aacgacggcc agtgaattgt aatacgactc actata
//
