LOCUS       sequence-218841-        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"
     primer_bind     27..49
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     41..58
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     42..58
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     65..84
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        65..83
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     116..132
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     116..132
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    1099..1666
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1116..1668
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1260..1277)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1487..1506
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1680..2381
                     /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(1813..1831)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2081..2100
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2275..2298
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(2416..2433)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2422..2646
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(2647..2680)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(3713..3731)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3752..3768)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3752..3768)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3765..3787)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3776..3792
                     /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(3800..3806,3807..3824,3825..3830))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3845..3866
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3983..4000)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4154..4742)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4234..4253)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4913..5704,5705..5773))
                     /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     5536..5555
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5774..5878)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5904..6359)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6041..6062)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6253..6272
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga
       61 attgtaatac gactcactat agggcgaatt ggagctccac cgcggtggcg gccgctctag
      121 aactagtgga tccgacacgc tcccctgact tgcgccccgc tccctctttc tttgcagcaa
      181 tgcctcctgc accaccaact gcttagcacc cctggccaag gtcatccatg acaactttgg
      241 tatcgtggaa ggactcatgg tatgagagct ggggaatggg actgaggctc ccacctttct
      301 catccaagac tggctcctcc ctgccggggc tgcgtgcaac cctggggttg ggggttctgg
      361 ggactggctt tcccataatt tcctttcaag gtggggaggg aggtagaggg gtgatgtggg
      421 gagtacgctg cagggcctca ctccttttgc agaccacagt ccatgccatc actgccaccc
      481 agaagactgt ggatggcccc tccgggaaac tgtggcgtga tggccgcggg gctctccaga
      541 acatcatccc tgcctctact ggcgctgcca aggctgtggg caaggtcatc cctgagctga
      601 acgggaagct cactggcatg gccttccgtg tccccactgc caacgtgtca gtggtggacc
      661 tgacctgccg tctagaaaaa cctgccaaat atgatgacat caagaaggtg gtgaagcagg
      721 cgtcggaggg ccccctcaag ggcatcctgg gctacactga gcaccaggtg gtctcctctg
      781 acttcaacag cgacacccac tcctccacct tcgacgctgg ggctggcatt gccctcaacg
      841 accactttgt caagctcatt tcctggtatg tggctggggc cagagactgg ctcttaaaaa
      901 gtgcagggtc tggcgccctc tggtggctgg ctcagaaaaa gggccctgac aactcttttc
      961 atcttctagg tatgacaacg aatttggcta cagcaacagg gtggtggacc tcatggccca
     1021 catggcctcc aaggagtaag acccctggac caccagcccc agcaagagca caagaggaac
     1081 aattgacgcg tgctcctctc cctccccccc ccntaacgtt actggccgaa gccgcttgga
     1141 ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa
     1201 tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc
     1261 tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc
     1321 ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg
     1381 cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca
     1441 accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag
     1501 cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct
     1561 ggggcctcgg tacacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc
     1621 cccgaaccac ggggacgtgg ttttcctttg aaaaacacga tgataatatg gccacaacca
     1681 tggtgagcaa gggcgaggag gtcatcaaag agttcatgcg cttcaaggtg cgcatggagg
     1741 gctccatgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     1801 gcacccagac cgccaagctg aaggtgacca agggcggccc cctgcccttc gcctgggaca
     1861 tcctgtcccc ccagttcatg tacggctcca aggcgtacgt gaagcacccc gccgacatcc
     1921 ccgattacaa gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     1981 aggacggcgg tctggtgacc gtgacccagg actcctccct gcaggacggc acgctgatct
     2041 acaaggtgaa gatgcgcggc accaacttcc cccccgacgg ccccgtaatg cagaagaaga
     2101 ccatgggctg ggaggcctcc accgagcgcc tgtacccccg cgacggcgtg ctgaagggcg
     2161 agatccacca ggccctgaag ctgaaggacg gcggccacta cctggtggag ttcaagacca
     2221 tctacatggc caagaagccc gtgcaactgc ccggctacta ctacgtggac accaagctgg
     2281 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgagcgc tccgagggcc
     2341 gccaccacct gttcctgtac ggcatggacg agctgtacaa gtaaatgcat cgatgatcta
     2401 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gctatctgtt gtttgcccct
     2461 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     2521 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     2581 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     2641 ctatggataa cttcgtatag catacattat acgaagttat cggaaccctt aatgtcgacc
     2701 cctgccacac tcagtccccc accacactga atctcccctc ctcacagttg ccatgtagac
     2761 cccttgaaga ggggaggggc ctagggagcc gcaccttgtc atgtaccatc aataaagtac
     2821 cctgtgctca accagttact tgtcctgtct tattctaggg tctggggcag aggggaggga
     2881 agctgggctt gtgtcaaggt gagacattct tgctggggag ggacctggta tgttctcctc
     2941 agactgaggg tagggcctcc aaacagcctt gcttgcttcg agaaccattt gcttcccgct
     3001 cagacgtctt gagtgctaca ggaagctggc accactactt cagagaacaa ggccttttcc
     3061 tctcctcgct ccagtcctag gctatctgct gttggccaaa catggaagaa gctattctgt
     3121 gggcggcccc agggaggctg acaggtggag gaagtcaggg ctcgcactgg gctctgacgc
     3181 tgactggtta gtggagctca gcctggagct gagctgcagc gggcaattcc agcttggcct
     3241 ccgcagctgt gaggtcttga gcacgtgctc tattgctttc tgtgccctcg tgtcttatct
     3301 gaggacatcg tggccagccc ctaaggtctt caagcaggat tcatctaggt aaaccaagta
     3361 cctaaaacca tgcccaaggc ggtaaggact atataatgtt taaaaatcgg taaaaatgcc
     3421 cacctcgcat agttttgagg aagatgaact gagatgtgtc agggtgactt atttccatca
     3481 tcgtccttag gggagcttgg gtaggggcaa ggcgtgtagc tgggacctag gtccagaccc
     3541 ctggctctgc cactgaacgg ctcaggtgct ttgggcagtt actccccggc ctcactttgc
     3601 acgtgtgctt acctagtgga gacaaaagta catacctcgg tagagcgcgc acgcctgtaa
     3661 ccccaggtta actctctaac ctcgaggggg ggcccggtac cagcttttgt tccctttagt
     3721 gagggttaat ttcgagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     3781 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     3841 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3901 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3961 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4021 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4081 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4141 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4201 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4261 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4321 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4381 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4441 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4501 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4561 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     4621 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4681 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4741 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4801 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4861 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4921 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4981 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5041 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5101 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5161 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5221 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5281 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5341 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5401 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5461 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5521 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5581 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5641 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5701 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5761 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5821 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     5881 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
     5941 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     6001 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     6061 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     6121 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     6181 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     6241 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     6301 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     6361 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     6421 attacgccag ctggcgaaag ggggatgtgc tgcaaggcg
//
