LOCUS       sequence-223512-        6459 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  donor to insert IRES-dTomato at the GAPDH locus in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6459)
  AUTHORS   Iyer S, Mir A, Vega-Badillo J, Roscoe BP, Ibraheim R, Zhu LJ, Lee J,
            Liu P, Luk K, Mintzer E, Guo D, Soares de Brito J, Emerson CP Jr, 
            Zamore PD, Sontheimer EJ, Wolfe SA
  TITLE     Efficient Homology-Directed Repair with Circular Single-Stranded DNA
            Donors.
  JOURNAL   CRISPR J. 2022 Oct;5(5):685-701. doi: 10.1089/crispr.2022.0058. Epub
            2022 Sep 7.
  PUBMED    36070530
REFERENCE   2  (bases 1 to 6459)
  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"; doi: 
            "10.1089/crispr.2022.0058"; journalName: "CRISPR J"; date: 
            "2022-10"; volume: "5"; issue: "5"; pages: "685-701"
COMMENT     Sequence Label: pBS-f1(+)-GAPDH-IRES-dTomato
FEATURES             Location/Qualifiers
     source          1..6459
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    944..1511
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    961..1513
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1105..1122)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1332..1351
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1525..2226
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(1658..1676)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1926..1945
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2120..2143
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(2261..2278)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2267..2491
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(2492..2525)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(3558..3576)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3597..3613)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3597..3613)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3610..3632)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3621..3637
                     /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(3645..3651,3652..3669,3670..3675))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3690..3711
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3828..3845)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3999..4587)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4079..4098)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4758..5549,5550..5618))
                     /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     5381..5400
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5619..5723)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5749..6204)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5886..5907)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6098..6117
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6331..6353
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6345..6362
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6346..6362
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6369..6388
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6369..6387
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     6420..6436
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6420..6436
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
ORIGIN
        1 cccgctccct ctttctttgc agcaatgcct cctgcaccac caactgctta gcacccctgg
       61 ccaaggtcat ccatgacaac tttggtatcg tggaaggact catggtatga gagctgggga
      121 atgggactga ggctcccacc tttctcatcc aagactggct cctccctgcc ggggctgcgt
      181 gcaaccctgg ggttgggggt tctggggact ggctttccca taatttcctt tcaaggtggg
      241 gagggaggta gaggggtgat gtggggagta cgctgcaggg cctcactcct tttgcagacc
      301 acagtccatg ccatcactgc cacccagaag actgtggatg gcccctccgg gaaactgtgg
      361 cgtgatggcc gcggggctct ccagaacatc atccctgcct ctactggcgc tgccaaggct
      421 gtgggcaagg tcatccctga gctgaacggg aagctcactg gcatggcctt ccgtgtcccc
      481 actgccaacg tgtcagtggt ggacctgacc tgccgtctag aaaaacctgc caaatatgat
      541 gacatcaaga aggtggtgaa gcaggcgtcg gagggccccc tcaagggcat cctgggctac
      601 actgagcacc aggtggtctc ctctgacttc aacagcgaca cccactcctc caccttcgac
      661 gctggggctg gcattgccct caacgaccac tttgtcaagc tcatttcctg gtatgtggct
      721 ggggccagag actggctctt aaaaagtgca gggtctggcg ccctctggtg gctggctcag
      781 aaaaagggcc ctgacaactc ttttcatctt ctaggtatga caacgaattt ggctacagca
      841 acagggtggt ggacctcatg gcccacatgg cctccaagga gtaagacccc tggaccacca
      901 gccccagcaa gagcacaaga ggaacaattg acgcgtgctc ctctccctcc ccccccccta
      961 acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt
     1021 ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga
     1081 cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg
     1141 tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt
     1201 gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat
     1261 aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg
     1321 aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg
     1381 taccccattg tatgggatct gatctggggc ctcggtacac atgctttaca tgtgtttagt
     1441 cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa
     1501 cacgatgata atatggccac aaccatggtg agcaagggcg aggaggtcat caaagagttc
     1561 atgcgcttca aggtgcgcat ggagggctcc atgaacggcc acgagttcga gatcgagggc
     1621 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggc
     1681 ggccccctgc ccttcgcctg ggacatcctg tccccccagt tcatgtacgg ctccaaggcg
     1741 tacgtgaagc accccgccga catccccgat tacaagaagc tgtccttccc cgagggcttc
     1801 aagtgggagc gcgtgatgaa cttcgaggac ggcggtctgg tgaccgtgac ccaggactcc
     1861 tccctgcagg acggcacgct gatctacaag gtgaagatgc gcggcaccaa cttccccccc
     1921 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctccaccga gcgcctgtac
     1981 ccccgcgacg gcgtgctgaa gggcgagatc caccaggccc tgaagctgaa ggacggcggc
     2041 cactacctgg tggagttcaa gaccatctac atggccaaga agcccgtgca actgcccggc
     2101 tactactacg tggacaccaa gctggacatc acctcccaca acgaggacta caccatcgtg
     2161 gaacagtacg agcgctccga gggccgccac cacctgttcc tgtacggcat ggacgagctg
     2221 tacaagtaaa tgcatcgatg atctagagct cgctgatcag cctcgactgt gccttctagt
     2281 tgccagctat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     2341 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     2401 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     2461 aggcatgctg gggatgcggt gggctctatg gataacttcg tatagcatac attatacgaa
     2521 gttatcggaa cccttaatgt cgacccctgc cacactcagt cccccaccac actgaatctc
     2581 ccctcctcac agttgccatg tagacccctt gaagagggga ggggcctagg gagccgcacc
     2641 ttgtcatgta ccatcaataa agtaccctgt gctcaaccag ttacttgtcc tgtcttattc
     2701 tagggtctgg ggcagagggg agggaagctg ggcttgtgtc aaggtgagac attcttgctg
     2761 gggagggacc tggtatgttc tcctcagact gagggtaggg cctccaaaca gccttgcttg
     2821 cttcgagaac catttgcttc ccgctcagac gtcttgagtg ctacaggaag ctggcaccac
     2881 tacttcagag aacaaggcct tttcctctcc tcgctccagt cctaggctat ctgctgttgg
     2941 ccaaacatgg aagaagctat tctgtgggcg gccccaggga ggctgacagg tggaggaagt
     3001 cagggctcgc actgggctct gacgctgact ggttagtgga gctcagcctg gagctgagct
     3061 gcagcgggca attccagctt ggcctccgca gctgtgaggt cttgagcacg tgctctattg
     3121 ctttctgtgc cctcgtgtct tatctgagga catcgtggcc agcccctaag gtcttcaagc
     3181 aggattcatc taggtaaacc aagtacctaa aaccatgccc aaggcggtaa ggactatata
     3241 atgtttaaaa atcggtaaaa atgcccacct cgcatagttt tgaggaagat gaactgagat
     3301 gtgtcagggt gacttatttc catcatcgtc cttaggggag cttgggtagg ggcaaggcgt
     3361 gtagctggga cctaggtcca gacccctggc tctgccactg aacggctcag gtgctttggg
     3421 cagttactcc ccggcctcac tttgcacgtg tgcttaccta gtggagacaa aagtacatac
     3481 ctcggtagag cgcgcacgcc tgtaacccca ggttaactct ctaacctcga gggggggccc
     3541 ggtaccagct tttgttccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca
     3601 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3661 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3721 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3781 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     3841 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3901 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3961 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4021 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4081 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4141 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4201 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4261 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4321 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4381 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4441 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4501 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4561 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4621 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4681 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4741 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4801 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4861 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4921 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4981 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5041 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5101 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5161 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5221 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5281 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5341 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5401 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5461 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5521 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5581 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5641 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5701 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg
     5761 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     5821 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     5881 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     5941 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     6001 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     6061 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     6121 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     6181 ttaatgcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag
     6241 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     6301 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     6361 gtgaattgta atacgactca ctatagggcg aattggagct ccaccgcggt ggcggccgct
     6421 ctagaactag tggatccgac acgctcccct gacttgcgc
//
