LOCUS       sequence-255031-        4795 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor empty vector with floxed puromycin resistance gene expression 
            cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4795)
  AUTHORS   Chen Y, Cao J, Xiong M, Petersen AJ, Dong Y, Tao Y, Huang CT, Du Z, 
            Zhang SC
  TITLE     Engineering Human Stem Cell Lines with Inducible Gene Knockout using
            CRISPR/Cas9.
  JOURNAL   Cell Stem Cell. 2015 Jul 1. pii: S1934-5909(15)00261-1. doi: 
            10.1016/j.stem.2015.06.001.
  PUBMED    26145478
REFERENCE   2  (bases 1 to 4795)
  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: "Cell Stem 
            Cell. 2015 Jul 1. pii: S1934-5909(15)00261-1. doi: 
            10.1016/j.stem.2015.06.001."
COMMENT     Sequence Label: PL552
FEATURES             Location/Qualifiers
     source          1..4795
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    88..121
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        182..681
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(217..240)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     614..633
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             721..1320
                     /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(721..740)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     protein_bind    1877..1910
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1936..1952)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1936..1952)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1984..2003)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1985..2003)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2010..2027)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2010..2026)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2019..2041)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2167..2622)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2304..2325)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2516..2535
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2649..2753
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2754..2822,2823..3614)
                     /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(2972..2991)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3785..4373
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4274..4293
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4527..4544
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4661..4682
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4697..4702,4703..4720,4721..4727)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4735..4751
                     /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     4740..4762
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4759..4775
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4759..4775
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 aattaaccct cactaaaggg aacaaaagct ggtaccgggc cccccctcga ggtcgacgaa
       61 ttccgatcat attcaataac ccttaatata acttcgtata atgtatgcta tacgaagtta
      121 ttaggtctga agaggagttt acgtccagcc aagcttagga tctcgacctc gaaattctac
      181 cgggtagggg aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg
      241 ggcacttggc gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg
      301 cgccaaccga ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc
      361 aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca
      421 cgtctcacta gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt
      481 tggggcagcg gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg
      541 ggtgggtccg ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc
      601 tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct
      661 tcctcatctc cgggcctttc gacctgcatc catctagatc tcgagcagct gaagcttacc
      721 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
      781 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
      841 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
      901 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
      961 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     1021 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     1081 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
     1141 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     1201 gagacctccg cgccccacaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     1261 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga
     1321 cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccca tgcatcgatg
     1381 atatcagatc cccgggatgc agaaattgat gatctattaa acaataaaga tgtccactaa
     1441 aatggaagtt tttcctgtca tactttgtta agaagggtga gaacagagta cctacatttt
     1501 gaatggaagg attggagcta cgggggtggg ggtggggtgg gattagataa atgcctgctc
     1561 tttactgaag gctctttact attgctttat gataatgttt catagttgga tatcataatt
     1621 taaacaagca aaaccaaatt aagggccagc tcattcctcc cactcatgat ctatagatct
     1681 atagatctct cgtgggatca ttgtttttct cttgattccc actttgtggt tctaagtact
     1741 gtggtttcca aatgtgtcag tttcatagcc tgaagaacga gatcagcagc ctctgttcca
     1801 catacacttc attctcagta ttgttttgcc aagttctaat tccatcagaa gctggtcgag
     1861 atccggaacc cttaatataa cttcgtataa tgtatgctat acgaagttat taggtccctc
     1921 gaagaggttc actaggatcc actagttcta gagcggccgc caccgcggtg gagctccaat
     1981 tcgccctata gtgagtcgta ttacaattca ctggccgtcg ttttacaacg tcgtgactgg
     2041 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     2101 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     2161 gaatggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     2221 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     2281 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     2341 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     2401 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     2461 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     2521 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     2581 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg
     2641 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     2701 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     2761 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     2821 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     2881 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     2941 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     3001 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     3061 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     3121 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     3181 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     3241 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     3301 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     3361 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     3421 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     3481 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     3541 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     3601 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     3661 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     3721 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3781 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3841 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3901 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     3961 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     4021 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     4081 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     4141 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     4201 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     4261 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     4321 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     4381 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     4441 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     4501 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     4561 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     4621 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     4681 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     4741 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctcga
//
