LOCUS       sequence-223514-        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     64..86
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     78..95
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     79..95
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     102..121
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        102..120
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     153..169
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     153..169
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    1136..1703
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1153..1705
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1297..1314)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1524..1543
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1717..2418
                     /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(1850..1868)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2118..2137
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2312..2335
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(2453..2470)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2459..2683
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(2684..2717)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(3750..3768)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3789..3805)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3789..3805)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3802..3824)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3813..3829
                     /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(3837..3843,3844..3861,3862..3867))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3882..3903
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4020..4037)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4191..4779)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4271..4290)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4950..5741,5742..5810))
                     /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     5573..5592
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5811..5915)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5941..6396
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6028..6047)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6238..6259
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
       61 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac tcactatagg
      121 gcgaattgga gctccaccgc ggtggcggcc gctctagaac tagtggatcc gacacgctcc
      181 cctgacttgc gccccgctcc ctctttcttt gcagcaatgc ctcctgcacc accaactgct
      241 tagcacccct ggccaaggtc atccatgaca actttggtat cgtggaagga ctcatggtat
      301 gagagctggg gaatgggact gaggctccca cctttctcat ccaagactgg ctcctccctg
      361 ccggggctgc gtgcaaccct ggggttgggg gttctgggga ctggctttcc cataatttcc
      421 tttcaaggtg gggagggagg tagaggggtg atgtggggag tacgctgcag ggcctcactc
      481 cttttgcaga ccacagtcca tgccatcact gccacccaga agactgtgga tggcccctcc
      541 gggaaactgt ggcgtgatgg ccgcggggct ctccagaaca tcatccctgc ctctactggc
      601 gctgccaagg ctgtgggcaa ggtcatccct gagctgaacg ggaagctcac tggcatggcc
      661 ttccgtgtcc ccactgccaa cgtgtcagtg gtggacctga cctgccgtct agaaaaacct
      721 gccaaatatg atgacatcaa gaaggtggtg aagcaggcgt cggagggccc cctcaagggc
      781 atcctgggct acactgagca ccaggtggtc tcctctgact tcaacagcga cacccactcc
      841 tccaccttcg acgctggggc tggcattgcc ctcaacgacc actttgtcaa gctcatttcc
      901 tggtatgtgg ctggggccag agactggctc ttaaaaagtg cagggtctgg cgccctctgg
      961 tggctggctc agaaaaaggg ccctgacaac tcttttcatc ttctaggtat gacaacgaat
     1021 ttggctacag caacagggtg gtggacctca tggcccacat ggcctccaag gagtaagacc
     1081 cctggaccac cagccccagc aagagcacaa gaggaacaat tgacgcgtgc tcctctccct
     1141 cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct
     1201 atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc
     1261 ctgtcttctt tacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc
     1321 tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg
     1381 tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa
     1441 agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt
     1501 ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg
     1561 atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtac acatgcttta
     1621 catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt
     1681 tcctttgaaa aacacgatga taatatggcc acaaccatgg tgagcaaggg cgaggaggtc
     1741 atcaaagagt tcatgcgctt caaggtgcgc atggagggct ccatgaacgg ccacgagttc
     1801 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag
     1861 gtgaccaagg gcggccccct gcccttcgcc tgggacatcc tgtcccccca gttcatgtac
     1921 ggctccaagg cgtacgtgaa gcaccccgcc gacatccccg attacaagaa gctgtccttc
     1981 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggtct ggtgaccgtg
     2041 acccaggact cctccctgca ggacggcacg ctgatctaca aggtgaagat gcgcggcacc
     2101 aacttccccc ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctccacc
     2161 gagcgcctgt acccccgcga cggcgtgctg aagggcgaga tccaccaggc cctgaagctg
     2221 aaggacggcg gccactacct ggtggagttc aagaccatct acatggccaa gaagcccgtg
     2281 caactgcccg gctactacta cgtggacacc aagctggaca tcacctccca caacgaggac
     2341 tacaccatcg tggaacagta cgagcgctcc gagggccgcc accacctgtt cctgtacggc
     2401 atggacgagc tgtacaagta aatgcatcga tgatctagag ctcgctgatc agcctcgact
     2461 gtgccttcta gttgccagct atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     2521 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     2581 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     2641 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggataactt cgtatagcat
     2701 acattatacg aagttatcgg aacccttaat gtcgacccct gccacactca gtcccccacc
     2761 acactgaatc tcccctcctc acagttgcca tgtagacccc ttgaagaggg gaggggccta
     2821 gggagccgca ccttgtcatg taccatcaat aaagtaccct gtgctcaacc agttacttgt
     2881 cctgtcttat tctagggtct ggggcagagg ggagggaagc tgggcttgtg tcaaggtgag
     2941 acattcttgc tggggaggga cctggtatgt tctcctcaga ctgagggtag ggcctccaaa
     3001 cagccttgct tgcttcgaga accatttgct tcccgctcag acgtcttgag tgctacagga
     3061 agctggcacc actacttcag agaacaaggc cttttcctct cctcgctcca gtcctaggct
     3121 atctgctgtt ggccaaacat ggaagaagct attctgtggg cggccccagg gaggctgaca
     3181 ggtggaggaa gtcagggctc gcactgggct ctgacgctga ctggttagtg gagctcagcc
     3241 tggagctgag ctgcagcggg caattccagc ttggcctccg cagctgtgag gtcttgagca
     3301 cgtgctctat tgctttctgt gccctcgtgt cttatatgag gacatcgtgg ccagccccta
     3361 aggtcttcaa gcaggattca tctaggtaaa ccaagtacct aaaaccatgc ccaaggcggt
     3421 aaggactata taatgtttaa aaatcggtaa aaatgcccac ctcgcatagt tttgaggaag
     3481 atgaactgag atgtgtcagg gtgacttatt tccatcatcg tccttagggg agcttgggta
     3541 ggggcaaggc gtgtagctgg gacctaggtc cagacccctg gctctgccac tgaacggctc
     3601 aggtgctttg ggcagttact ccccggcctc actttgcacg tgtgcttacc tagtggagac
     3661 aaaagtacat acctcggtag agcgcgcacg cctgtaaccc caggttaact ctctaacctc
     3721 gagggggggc ccggtaccag cttttgttcc ctttagtgag ggttaatttc gagcttggcg
     3781 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     3841 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     3901 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3961 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     4021 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4081 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4141 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4201 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4261 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4321 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4381 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4441 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4501 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4561 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4621 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4681 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4741 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4801 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     4861 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4921 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4981 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     5041 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     5101 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     5161 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     5221 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     5281 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     5341 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5401 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5461 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5521 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5581 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5641 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5701 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5761 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     5821 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     5881 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5941 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     6001 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     6061 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     6121 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     6181 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     6241 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     6301 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     6361 acgcgaattt taacaaaata ttaacgctta caatttccca ttcgccattc aggctgcgca
     6421 actgttggga agggcgatcg gtgcgggcct cttcgctat
//
