LOCUS       sequence-319266-        4747 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor empty vector with puromycin resistance gene expression 
            cassette flanked by FRT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4747)
  AUTHORS   Xiong M, Tao Y, Gao Q, Feng B, Yan W, Zhou Y, Kotsonis TA, Yuan T, 
            You Z, Wu Z, Xi J, Haberman A, Graham J, Block J, Zhou W, Chen Y, 
            Zhang SC
  TITLE     Human Stem Cell-Derived Neurons Repair Circuits and Restore Neural 
            Function.
  JOURNAL   Cell Stem Cell. 2020 Sep 16. pii: S1934-5909(20)30410-0. doi: 
            10.1016/j.stem.2020.08.014.
  PUBMED    32966778
REFERENCE   2  (bases 1 to 4747)
  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. 2020 Sep 16. pii: S1934-5909(20)30410-0. doi: 
            10.1016/j.stem.2020.08.014."
COMMENT     Sequence Label: PL652
FEATURES             Location/Qualifiers
     source          1..4747
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..500
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(36..59)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     433..452
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             540..1139
                     /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(540..559)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     protein_bind    1696..1729
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(1731..1747)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1731..1747)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1779..1798)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1780..1798)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1805..1822)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1805..1821)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1814..1836)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(1962..2417)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2099..2120)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2311..2330
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2444..2548
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2549..2617,2618..3409)
                     /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(2767..2786)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3580..4168
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4069..4088
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4322..4339
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4456..4477
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4492..4497,4498..4515,4516..4522)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4530..4546
                     /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     4535..4557
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4554..4570
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4554..4570
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        4591..4609
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    4654..4687
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
ORIGIN
        1 gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg
       61 gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc
      121 gccaaccgac tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca
      181 ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac
      241 gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt
      301 ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg
      361 gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct
      421 ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt
      481 cctcatctcc gggcctttcg acctgcatcc atctagatct cgagcagctg aagcttacca
      541 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac
      601 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc
      661 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
      721 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga
      781 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
      841 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc
      901 ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca
      961 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg
     1021 agacctccgc gccccacaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     1081 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgac
     1141 gcccgcccca cgacccgcag cgcccgaccg aaaggagcgc acgaccccat gcatcgatga
     1201 tatcagatcc ccgggatgca gaaattgatg atctattaaa caataaagat gtccactaaa
     1261 atggaagttt ttcctgtcat actttgttaa gaagggtgag aacagagtac ctacattttg
     1321 aatggaagga ttggagctac gggggtgggg gtggggtggg attagataaa tgcctgctct
     1381 ttactgaagg ctctttacta ttgctttatg ataatgtttc atagttggat atcataattt
     1441 aaacaagcaa aaccaaatta agggccagct cattcctccc actcatgatc tatagatcta
     1501 tagatctctc gtgggatcat tgtttttctc ttgattccca ctttgtggtt ctaagtactg
     1561 tggtttccaa atgtgtcagt ttcatagcct gaagaacgag atcagcagcc tctgttccac
     1621 atacacttca ttctcagtat tgttttgcca agttctaatt ccatcagaag ctggtcgaga
     1681 tccggaaccc ttaatgaagt tcctattctc tagaaagtat aggaacttcg gatccactag
     1741 ttctagagcg gccgccaccg cggtggagct ccaattcgcc ctatagtgag tcgtattaca
     1801 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     1861 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     1921 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gaaattgtaa gcgttaatat
     1981 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     2041 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     2101 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     2161 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     2221 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     2281 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     2341 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     2401 gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     2461 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     2521 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     2581 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     2641 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     2701 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     2761 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     2821 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     2881 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     2941 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     3001 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     3061 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     3121 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     3181 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     3241 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     3301 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     3361 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     3421 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     3481 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     3541 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3601 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3661 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3721 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3781 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3841 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3901 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3961 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4021 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4081 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4141 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     4201 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     4261 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4321 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     4381 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     4441 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     4501 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     4561 cagctatgac catgattacg ccaagctcga aattaaccct cactaaaggg aacaaaagct
     4621 ggtaccgggc cccccctcga ggtcgacgaa ttcgaagttc ctattctcta gaaagtatag
     4681 gaacttctag gtctgaagag gagtttacgt ccagccaagc ttaggatctc gacctcgaaa
     4741 ttctacc
//
