LOCUS       sequence-223536-        5918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  donor to insert dTomato at the TOMM20 locus in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5918)
  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 5918)
  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(-)-TOMM20-dTomato
FEATURES             Location/Qualifiers
     source          1..5918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     87..109
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     101..118
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     102..118
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     125..144
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        125..143
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     176..192
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     176..192
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             1239..1937
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGL
                     VTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQ
                     ALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHH
                     LFLYGMDELYK"
     primer_bind     complement(1369..1387)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1637..1656
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1831..1854
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     promoter        complement(3232..3250)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3271..3287)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3271..3287)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3284..3306)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3295..3311
                     /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(3319..3325,3326..3343,3344..3349))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3364..3385
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3502..3519)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3673..4261)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3753..3772)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4432..5223,5224..5292))
                     /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     5055..5074
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5293..5397)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5423..5878
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5510..5529)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5720..5741
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
       61 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga
      121 attgtaatac gactcactat agggcgaatt ggagctccac cgcggtggcg gccgctctag
      181 aactagtgga tccgacgcgt attgggatga tgataggaag tatttacaga actttatagt
      241 tagtaactga ctggttaatt tttcaaactg atttttactc aactgaatta gaaaaggact
      301 ggaaagaaag taaagatccc aacgacttga gggaacaagt tggacaacca aggactttgt
      361 ctaaattgtt tttatttaga ctaatgtggt tctagttcta gaggattcat actggaatca
      421 tcggtttaat attacgctat ttgaaaggca gcatagtata gtacttttgg aaaattggcc
      481 tgagggtgat gtcttttgga atatttgtga attcactatg aagcctaatt ccttaaaaat
      541 gacctccttc actcaattat cagtgttctt ggtttgcctg ggagtgaaaa gagatcttaa
      601 aatctttttg gttttagtta cataattgac tgatgtaata ttatgtaatg atggctgtac
      661 acagtgtctc atgccctata atcctagcac tcatttgagc tcaggagttc aaggccagcc
      721 tgggcaacat ggtaaaaccc tgtctgcacc aaaaataaaa aaaaaattag ccgggcatgg
      781 tggcatgagt ctgtggttcc agctactcag gaggctgagg tgggggagga tcgcttgagc
      841 ccaggagtca gaggttgcag tgagctgaga ttgtgctact gcactccagc ctgggttaca
      901 gagaccccat ctgaattaaa aacatatata atgtaatgat ctgcctcctt tgttaacttg
      961 acttttgaaa tgggattgtc agtagtatga tcattgtttt cttggatgcc gactgtgtgt
     1021 aaagtgttac attttgaatt aaatgtcaga atgggtgaac tttactaaga ttcaattctt
     1081 tgaatacaaa gagcatttta ttttgaagtt agaatactaa ttaaatgctt atgacacttt
     1141 aaaaaattat tttttttttc tttcagagaa ttgtaagtgc acagtccttg gctgaagatg
     1201 atgtggaagg cggtagcggg gatccaccgg tcgccaccgt gagcaagggc gaggaggtca
     1261 tcaaagagtt catgcgcttc aaggtgcgca tggagggctc catgaacggc cacgagttcg
     1321 agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg
     1381 tgaccaaggg cggccccctg cccttcgcct gggacatcct gtccccccag ttcatgtacg
     1441 gctccaaggc gtacgtgaag caccccgccg acatccccga ttacaagaag ctgtccttcc
     1501 ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggtctg gtgaccgtga
     1561 cccaggactc ctccctgcag gacggcacgc tgatctacaa ggtgaagatg cgcggcacca
     1621 acttcccccc cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctccaccg
     1681 agcgcctgta cccccgcgac ggcgtgctga agggcgagat ccaccaggcc ctgaagctga
     1741 aggacggcgg ccactacctg gtggagttca agaccatcta catggccaag aagcccgtgc
     1801 aactgcccgg ctactactac gtggacacca agctggacat cacctcccac aacgaggact
     1861 acaccatcgt ggaacagtac gagcgctccg agggccgcca ccacctgttc ctgtacggca
     1921 tggacgagct gtacaagtaa tgagaaacaa atgtcaacat aataaaatct cagttaaaaa
     1981 tattttaaaa attcttggta gttgagcagc tctggggtaa taagggcaaa tatgcttgtt
     2041 atgaactaca ctgaaatcta ccaaagttaa tgtttacttt gtgtagatcc atttgtctat
     2101 tttatttatt tttcccagtg aaaagtgtat tttgatagag aacttttcat tctataaata
     2161 cactatgagt tactaaaata tcatggattt tgtttattcc tgaaacatag ttacatagtt
     2221 aaactgtaca tatgacatgg cttatgttaa aaatacccag tgctcagttt tgaaagatag
     2281 gcaaaaaaaa aaagtatagg agaaactgaa gaatgtacac ttttttagag ggcacatttt
     2341 gctgtaaatc tggaaatttg atagacttga ctgtgtttgt gaaaactgag cattaaaggt
     2401 tttgattgat cctttctttc catttaatct ctgagacgta aatatgtgag gtgtgctgct
     2461 gtgctgggtt aacagcttcc ttccctttct gtgtagcagt cttgaaatgt tctgtttaaa
     2521 tcagtaggct taatgtgttc tgggtattta tctccttgta ttttaaatat atgtagttgc
     2581 aaatagcacc aggaattaga tttctgtaca cccctaatct agccttgtga gcttcgctag
     2641 ttaatgtgtg ctcactttcc ctccatttgt tacgtgagag aatgcgtctg ctgatcactg
     2701 aagtgtccct tttagcttct gattcattgg gttctgttgg gcatctttaa atccacctta
     2761 acctgaggaa tgtatgtggg caaccaggcc ctgcattttt ttatattctg aattttgcat
     2821 gcttgcctga cttagtattt ctgaattgat gtttttttta atggtataac tatcttgatt
     2881 ttcactgaaa ttatatggtt ctgtcactac tctgtaaatt aatccgaaac ttttaaggta
     2941 tcccaatggc gctttgagga agatgaactg agatgtgtca gggtgactta tttccatcat
     3001 cgtccttagg ggagcttggg taggggcaag gcgtgtagct gggacctagg tccagacccc
     3061 tggctctgcc actgaacggc tcaggtgctt tgggcagtta ctccccggcc tcactttgca
     3121 cgtgtgctta cctagtggag acaaaagtac atacctcggt agagcgcgca cgcctgtaac
     3181 cccaggttaa ctctctaacc tcgagggggg gcccggtacc agcttttgtt ccctttagtg
     3241 agggttaatt tcgagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     3301 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     3361 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     3421 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     3481 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     3541 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     3601 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     3661 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     3721 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     3781 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     3841 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     3901 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     3961 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4021 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4081 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4141 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4201 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4261 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4321 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     4381 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     4441 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     4501 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     4561 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     4621 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     4681 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     4741 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     4801 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     4861 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     4921 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     4981 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5041 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5101 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5161 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5221 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5281 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5341 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5401 atttccccga aaagtgccac ctacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     5461 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     5521 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     5581 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     5641 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     5701 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     5761 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     5821 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcc
     5881 cattcgccat tcaggctgcg caactgttgg gaagggcg
//
