LOCUS       sequence-223580-        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     44..66
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     58..75
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     59..75
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     82..101
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        82..100
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     133..149
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     133..149
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    1116..1683
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1133..1685
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1277..1294)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1504..1523
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(1697..1699,1700..1702,1703..2416)
                     /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(1697..1699,1700..1702,1703..2413)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1742..1763)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2003..2022)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2350..2371
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2448..2465)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2454..2678
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(2679..2712)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(3745..3763)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3784..3800)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3784..3800)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3797..3819)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3808..3824
                     /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(3832..3838,3839..3856,3857..3862))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3877..3898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4015..4032)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4186..4774)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4266..4285)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4945..5736,5737..5805))
                     /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     5568..5587
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5806..5910)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5936..6391
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6023..6042)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6233..6254
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
       61 aaaacgacgg ccagtgaatt gtaatacgac tcactatagg gcgaattgga gctccaccgc
      121 ggtggcggcc gctctagaac tagtggatcc gacacgctcc cctgacttgc gccccgctcc
      181 ctctttcttt gcagcaatgc ctcctgcacc accaactgct tagcacccct ggccaaggtc
      241 atccatgaca actttggtat cgtggaagga ctcatggtat gagagctggg gaatgggact
      301 gaggctccca cctttctcat ccaagactgg ctcctccctg ccggggctgc gtgcaaccct
      361 ggggttgggg gttctgggga ctggctttcc cataatttcc tttcaaggtg gggagggagg
      421 tagaggggtg atgtggggag tacgctgcag ggcctcactc cttttgcaga ccacagtcca
      481 tgccatcact gccacccaga agactgtgga tggcccctcc gggaaactgt ggcgtgatgg
      541 ccgcggggct ctccagaaca tcatccctgc ctctactggc gctgccaagg ctgtgggcaa
      601 ggtcatccct gagctgaacg ggaagctcac tggcatggcc ttccgtgtcc ccactgccaa
      661 cgtgtcagtg gtggacctga cctgccgtct agaaaaacct gccaaatatg atgacatcaa
      721 gaaggtggtg aagcaggcgt cggagggccc cctcaagggc atcctgggct acactgagca
      781 ccaggtggtc tcctctgact tcaacagcga cacccactcc tccaccttcg acgctggggc
      841 tggcattgcc ctcaacgacc actttgtcaa gctcatttcc tggtatgtgg ctggggccag
      901 agactggctc ttaaaaagtg cagggtctgg cgccctctgg tggctggctc agaaaaaggg
      961 ccctgacaac tcttttcatc ttctaggtat gacaacgaat ttggctacag caacagggtg
     1021 gtggacctca tggcccacat ggcctccaag gagtaagacc cctggaccac cagccccagc
     1081 aagagcacaa gaggaacaat tgacgcgtgc tcctctccct cccccccccc taacgttact
     1141 ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt ttccaccata
     1201 ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt gacgagcatt
     1261 cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa
     1321 gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag
     1381 cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt ataagataca
     1441 cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc
     1501 aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa ggtaccccat
     1561 tgtatgggat ctgatctggg gcctcggtac acatgcttta catgtgttta gtcgaggtta
     1621 aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa aacacgatga
     1681 taatatggcc acaaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     1741 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     1801 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     1861 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     1921 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     1981 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     2041 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     2101 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     2161 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     2221 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     2281 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     2341 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     2401 cgagctgtac aagtaaatgc atcgatgatc tagagctcgc tgatcagcct cgactgtgcc
     2461 ttctagttgc cagctatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2521 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2581 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2641 caatagcagg catgctgggg atgcggtggg ctctatggat aacttcgtat agcatacatt
     2701 atacgaagtt atcggaaccc ttaatgtcga cccctgccac actcagtccc ccaccacact
     2761 gaatctcccc tcctcacagt tgccatgtag accccttgaa gaggggaggg gcctagggag
     2821 ccgcaccttg tcatgtacca tcaataaagt accctgtgct caaccagtta cttgtcctgt
     2881 cttattctag ggtctggggc agaggggagg gaagctgggc ttgtgtcaag gtgagacatt
     2941 cttgctgggg agggacctgg tatgttctcc tcagactgag ggtagggcct ccaaacagcc
     3001 ttgcttgctt cgagaaccat ttgcttcccg ctcagacgtc ttgagtgcta caggaagctg
     3061 gcaccactac ttcagagaac aaggcctttt cctctcctcg ctccagtcct aggctatctg
     3121 ctgttggcca aacatggaag aagctattct gtgggcggcc ccagggaggc tgacaggtgg
     3181 aggaagtcag ggctcgcact gggctctgac gctgactggt tagtggagct cagcctggag
     3241 ctgagctgca gcgggcaatt ccagcttggc ctccgcagct gtgaggtctt gagcacgtgc
     3301 tctattgctt tctgtgccct cgtgtcttat ctgaggacat cgtggccagc ccctaaggtc
     3361 ttcaagcagg attcatctag gtaaaccaag tacctaaaac catgcccaag gcggtaagga
     3421 ctatataatg tttaaaaatc ggtaaaaatg cccacctcgc atagttttga ggaagatgaa
     3481 ctgagatgtg tcagggtgac ttatttccat catcgtcctt aggggagctt gggtaggggc
     3541 aaggcgtgta gctgggacct aggtccagac ccctggctct gccactgaac ggctcaggtg
     3601 ctttgggcag ttactccccg gcctcacttt gcacgtgtgc ttacctagtg gagacaaaag
     3661 tacatacctc ggtagagcgc gcacgcctgt aaccccaggt taactctcta acctcgaggg
     3721 ggggcccggt accagctttt gttcccttta gtgagggtta atttcgagct tggcgtaatc
     3781 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     3841 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     3901 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     3961 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4021 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4081 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4141 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4201 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4261 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4321 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4381 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4441 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4501 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4561 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4621 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4681 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4741 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     4801 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     4861 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     4921 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     4981 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5041 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5101 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5161 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5221 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5281 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     5341 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5401 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     5461 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5521 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     5581 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5641 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5701 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     5761 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     5821 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     5881 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctacgcg
     5941 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     6001 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     6061 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     6121 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     6181 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     6241 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     6301 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     6361 aattttaaca aaatattaac gcttacaatt tcccattcgc cattcaggct gcgcaactgt
     6421 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa aggg
//
