LOCUS       sequence-218843-        6474 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  donor to insert IRES-EGFP at the GAPDH locus in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6474)
  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 6474)
  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-EGFP
FEATURES             Location/Qualifiers
     source          1..6474
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     127..149
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     141..158
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     142..158
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     165..184
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        165..183
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     216..232
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     216..232
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    1199..1766
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1216..1768
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1360..1377)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1587..1606
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(1780..1782,1783..1785,1786..2499)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1780..1782,1783..1785,1786..2496)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1825..1846)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2086..2105)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2433..2454
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2531..2548)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2537..2761
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(2762..2795)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(3828..3846)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3867..3883)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3867..3883)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3880..3902)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3891..3907
                     /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(3915..3921,3922..3939,3940..3945))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3960..3981
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4098..4115)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4269..4857)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4349..4368)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5028..5819,5820..5888))
                     /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     5651..5670
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5889..5993)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6019..6447
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6106..6125)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6316..6337
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
       61 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
      121 ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac gactcactat
      181 agggcgaatt ggagctccac cgcggtggcg gccgctctag aactagtgga tccgacacgc
      241 tcccctgact tgcgccccgc tccctctttc tttgcagcaa tgcctcctgc accaccaact
      301 gcttagcacc cctggccaag gtcatccatg acaactttgg tatcgtggaa ggactcatgg
      361 tatgagagct ggggaatggg actgaggctc ccacctttct catccaagac tggctcctcc
      421 ctgccggggc tgcgtgcaac cctggggttg ggggttctgg ggactggctt tcccataatt
      481 tcctttcaag gtggggaggg aggtagaggg gtgatgtggg gagtacgctg cagggcctca
      541 ctccttttgc agaccacagt ccatgccatc actgccaccc agaagactgt ggatggcccc
      601 tccgggaaac tgtggcgtga tggccgcggg gctctccaga acatcatccc tgcctctact
      661 ggcgctgcca aggctgtggg caaggtcatc cctgagctga acgggaagct cactggcatg
      721 gccttccgtg tccccactgc caacgtgtca gtggtggacc tgacctgccg tctagaaaaa
      781 cctgccaaat atgatgacat caagaaggtg gtgaagcagg cgtcggaggg ccccctcaag
      841 ggcatcctgg gctacactga gcaccaggtg gtctcctctg acttcaacag cgacacccac
      901 tcctccacct tcgacgctgg ggctggcatt gccctcaacg accactttgt caagctcatt
      961 tcctggtatg tggctggggc cagagactgg ctcttaaaaa gtgcagggtc tggcgccctc
     1021 tggtggctgg ctcagaaaaa gggccctgac aactcttttc atcttctagg tatgacaacg
     1081 aatttggcta cagcaacagg gtggtggacc tcatggccca catggcctcc aaggagtaag
     1141 acccctggac caccagcccc agcaagagca caagaggaac aattgacgcg tgctcctctc
     1201 cctccccccc ccntaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg
     1261 tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg
     1321 gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag
     1381 gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt
     1441 ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc
     1501 aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga
     1561 gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga
     1621 aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tacacatgct
     1681 ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg
     1741 ttttcctttg aaaaacacga tgataatatg gccacaacca tggtgagcaa gggcgaggag
     1801 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1861 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     1921 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     1981 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     2041 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     2101 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     2161 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     2221 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     2281 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     2341 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     2401 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     2461 gccgggatca ctctcggcat ggacgagctg tacaagtaaa tgcatcgatg atctagagct
     2521 cgctgatcag cctcgactgt gccttctagt tgccagctat ctgttgtttg cccctccccc
     2581 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     2641 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     2701 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     2761 gataacttcg tatagcatac attatacgaa gttatcggaa cccttaatgt cgacccctgc
     2821 cacactcagt cccccaccac actgaatctc ccctcctcac agttgccatg tagacccctt
     2881 gaagagggga ggggcctagg gagccgcacc ttgtcatgta ccatcaataa agtaccctgt
     2941 gctcaaccag ttacttgtcc tgtcttattc tagggtctgg ggcagagggg agggaagctg
     3001 ggcttgtgtc aaggtgagac attcttgctg gggagggacc tggtatgttc tcctcagact
     3061 gagggtaggg cctccaaaca gccttgcttg cttcgagaac catttgcttc ccgctcagac
     3121 gtcttgagtg ctacaggaag ctggcaccac tacttcagag aacaaggcct tttcctctcc
     3181 tcgctccagt cctaggctat ctgctgttgg ccaaacatgg aagaagctat tctgtgggcg
     3241 gccccaggga ggctgacagg tggaggaagt cagggctcgc actgggctct gacgctgact
     3301 ggttagtgga gctcagcctg gagctgagct gcagcgggca attccagctt ggcctccgca
     3361 gctgtgaggt cttgagcacg tgctctattg ctttctgtgc cctcgtgtct tatctgagga
     3421 catcgtggcc agcccctaag gtcttcaagc aggattcatc taggtaaacc aagtacctaa
     3481 aaccatgccc aaggcggtaa ggactatata atgtttaaaa atcggtaaaa atgcccacct
     3541 cgcatagttt tgaggaagat gaactgagat gtgtcagggt gacttatttc catcatcgtc
     3601 cttaggggag cttgggtagg ggcaaggcgt gtagctggga cctaggtcca gacccctggc
     3661 tctgccactg aacggctcag gtgctttggg cagttactcc ccggcctcac tttgcacgtg
     3721 tgcttaccta gtggagacaa aagtacatac ctcggtagag cgcgcacgcc tgtaacccca
     3781 ggttaactct ctaacctcga gggggggccc ggtaccagct tttgttccct ttagtgaggg
     3841 ttaatttcga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     3901 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3961 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4021 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4081 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4141 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4201 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4261 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4321 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4381 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4441 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4501 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4561 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4621 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4681 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     4741 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4801 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4861 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4921 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4981 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5041 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     5101 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5161 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5221 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5281 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5341 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5401 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5461 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5521 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5581 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5641 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5701 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5761 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5821 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5881 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5941 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6001 ccccgaaaag tgccacctac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     6061 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     6121 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     6181 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     6241 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     6301 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     6361 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     6421 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca attt
//
