LOCUS       sequence-255697-        5161 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology dependent repair construct with positive selection 
            puromycin and negative selection viral thymidine kinase cassettes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5161)
  AUTHORS   Giacalone JC, Sharma TP, Burnight ER, Fingert JF, Mullins RF, Stone 
            EM, Tucker BA
  TITLE     CRISPR-Cas9-Based Genome Editing of Human Induced Pluripotent Stem 
            Cells.
  JOURNAL   Curr Protoc Stem Cell Biol. 2018 Feb 28;44:5B.7.1-5B.7.22. doi: 
            10.1002/cpsc.46.
  PUBMED    29512106
REFERENCE   2  (bases 1 to 5161)
  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: "Curr Protoc
            Stem Cell Biol."; date: "2018-02-28"; volume: "44:5B.7.1-5B.7.22. 
            doi"; pages: " 10.1002/cpsc.46"
COMMENT     Sequence Label: pHDR mPGK-puroTK
FEATURES             Location/Qualifiers
     source          1..5161
                     /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             506..1102
                     /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(506..525)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1002..1022
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1112..1168
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1169..2299
                     /codon_start=1
                     /product="herpes simplex virus thymidine kinase"
                     /label=HSV TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRLEQK
                     MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETIANIYTTQ
                     HRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDR
                     HPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQR
                     PGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAVPPQGAEPQSNAGPR
                     PHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDAL
                     LQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     polyA_signal    2300..2421
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2337..2356)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2391..2410
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2432..2465
                     /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(2490..2506)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2490..2506)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2503..2525)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2514..2530
                     /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(2538..2544,2545..2562,2563..2568))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2583..2604
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2721..2738)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2892..3480)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2972..2991)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3651..4442,4443..4511))
                     /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     4274..4293
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4512..4616)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4684..4702
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4740..4762)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4862..4881
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5075..5097
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5089..5106
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5090..5106
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5123..5156
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg
       61 gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc
      121 gccaaccggc 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 acaccatgac cgagtacaag cccacggtgc gcctcgccac
      541 ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
      601 cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
      661 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
      721 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
      781 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
      841 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc
      901 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
      961 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga
     1021 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     1081 catgacccgc aagcccggtg ccggaagcgg agctactaac ttcagcctgc tgaagcaggc
     1141 tggagacgtg gaggagaacc ctggacctat ggcttcgtac ccctgccatc aacacgcgtc
     1201 tgcgttcgac caggctgcgc gttctcgcgg ccatagcaac cgacgtacgg cgttgcgccc
     1261 tcgccggcag caagaagcca cggaagtccg cctggagcag aaaatgccca cgctactgcg
     1321 ggtttatata gacggtcctc acgggatggg gaaaaccacc accacgcaac tgctggtggc
     1381 cctgggttcg cgcgacgata tcgtctacgt acccgagccg atgacttact ggcaggtgct
     1441 gggggcttcc gagacaatcg cgaacatcta caccacacaa caccgcctcg accagggtga
     1501 gatatcggcc ggggacgcgg cggtggtaat gacaagcgcc cagataacaa tgggcatgcc
     1561 ttatgccgtg accgacgccg ttctggctcc tcatatcggg ggggaggctg ggagctcaca
     1621 tgccccgccc ccggccctca ccctcatctt cgaccgccat cccatcgccg ccctcctgtg
     1681 ctacccggcc gcgcgatacc ttatgggcag catgaccccc caggccgtgc tggcgttcgt
     1741 ggccctcatc ccgccgacct tgcccggcac aaacatcgtg ttgggggccc ttccggagga
     1801 cagacacatc gaccgcctgg ccaaacgcca gcgccccggc gagcggcttg acctggctat
     1861 gctggccgcg attcgccgcg tttacgggct gcttgccaat acggtgcggt atctgcaggg
     1921 cggcgggtcg tggcgggagg attggggaca gctttcgggg acggccgtgc cgccccaggg
     1981 tgccgagccc cagagcaacg cgggcccacg accccatatc ggggacacgt tatttaccct
     2041 gtttcgggcc cccgagttgc tggcccccaa cggcgacctg tacaacgtgt ttgcctgggc
     2101 cttggacgtc ttggccaaac gcctccgtcc catgcacgtc tttatcctgg attacgacca
     2161 atcgcccgcc ggctgccggg acgccctgct gcaacttacc tccgggatgg tccagaccca
     2221 cgtcaccacc cccggctcca taccgacgat ctgcgacctg gcgcgcacgt ttgcccggga
     2281 gatgggggag gctaactgaa acttgtttat tgcagcttat aatggttaca aataaagcaa
     2341 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     2401 caaactcatc aatgtatctt atcatgtctg gataacttcg tataatgtat gctatacgaa
     2461 gttatagatc tctcgagggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     2521 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     2581 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     2641 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     2701 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     2761 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     2821 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     2881 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     2941 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3001 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3061 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3121 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3181 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     3241 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3301 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     3361 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     3421 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     3481 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     3541 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     3601 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     3661 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     3721 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     3781 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     3841 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     3901 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     3961 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     4021 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     4081 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     4141 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     4201 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     4261 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     4321 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     4381 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     4441 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     4501 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     4561 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     4621 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     4681 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     4741 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     4801 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     4861 gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga
     4921 tgcgtaagga gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg
     4981 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     5041 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
     5101 gccagtgggc ggccgcgcta gcataacttc gtataatgta tgctatacga agttatctac
     5161 c
//
