LOCUS       sequence-223487-        5845 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  donor to insert EGFP at the SEC61B locus in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5845)
  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 5845)
  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(+)-SEC61B-mEGFP
FEATURES             Location/Qualifiers
     source          1..5845
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(993..995,996..998,999..1709)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1038..1059)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1299..1318)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1646..1667
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2968..2986)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3007..3023)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3007..3023)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3020..3042)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3031..3047
                     /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(3055..3061,3062..3079,3080..3085))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3100..3121
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3238..3255)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3409..3997)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3489..3508)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4168..4959,4960..5028))
                     /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     4791..4810
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5029..5133)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5159..5614)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5296..5317)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5508..5527
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5741..5763
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5755..5772
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5756..5772
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5779..5798
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5779..5797
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     join(5830..5845,1)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5830..5845,1)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
ORIGIN
        1 ccgggcccac actaaagtta gagaaccaca ggctcgctca caaccctgac ttctccatgt
       61 cagttccgat ctttgcgaac cgcagacagg gaaggtcttc tctcaggggt catgcccgcg
      121 gccgccctcc acggcgaggt ccgcactcgc gcagccggcc ccgcggccgc ctcacctggt
      181 cgcacactac cacgtcgaac tcctcgtcgg cgaggaacag cacgtagagc gccaggaaaa
      241 ccatgcgcac gtaggcgcag acggcggcgc cgcggccgcc ccagcccagg cctcgcggca
      301 gccagtcccc ggcacagcgc accggtagct cgcggctctc ggcgaaacag tggcccgggt
      361 cgtagtgcgc tgtccagatc ttcacgctac acccgcgcgc ctgcagcgcc agcgccgcgt
      421 ccaacaccag ccgctcagcg ccgcccacgc ccaggtctgg gtggaggaac agcaccgacg
      481 gcttgggaac cgagtcccgt tcccggccct gctcctccgc catggccctg gagccgcaac
      541 tgcaccccgc accctgatgg gggtcttctg cgcaagctcc gcgctcgtag ctcccagctg
      601 gccactgcgg gccgaccccg ccctgccgta cgtgcgtcag ttaggccaca tcagcgcaaa
      661 tctgtgaggg tctagtaact gcctgagaaa atatcttgtc tgaccccggt tatatttttc
      721 cttcggtagg gattggactt tctgaaggac gttgtgatcc aaaggaagga ggccggaggt
      781 ctctacttcc catacagcag gtaactaagt tgtctgtagc agactgtcta caggcatatc
      841 gtgagacgac ccaggcgtcc ctggggtcag agaggacctt gcctgcaagt ccgggggcgg
      901 ggcctgagtc agtctcgcca gctgccggtc tttcgggggc tccgtaactt tctatccgtc
      961 cgcgtcagcg ccttgccaca ctcatctcca atatggtgag caagggcgag gagctgttca
     1021 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     1081 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     1141 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     1201 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     1261 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     1321 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     1381 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     1441 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     1501 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     1561 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc aagctgagca
     1621 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     1681 tcactctcgg catggacgag ctgtacaaga gcggcctgag aagcatggta tggcggccct
     1741 tccatgatcc ccgcctctcc cagaagccct gactcctcct gctttgcgcc gtgcttttcc
     1801 tctgtagctc ccttgcttcc cccagcctcg ggtgtgggtg tctaggccgg ggttctgggg
     1861 caggcctgcc gcgctcaccc gtctgtctgc ttgtctccct ctacagcctg gtccgacccc
     1921 cagtggcact aacgtgggat cctcagggcg ctctcccagc aaagcagtgg ccgcccgggc
     1981 ggcgggatcc actgtccggc agaggtaagg aaccctgcag ttcgttcgct tccagactcg
     2041 gagataggac ccagaacctc gctgattctg gggtggagac cctagcatgt gaagattgac
     2101 aaaggcaaaa tgagcttcta gtgacgtggc cgtgggagta gttaaaggcc ttttgggagg
     2161 aaggcgacat tttttttctc gttgctcagt ttagggcact actcttaaaa aaggaaagtt
     2221 aacaaactgg aatagagtca gagataactt tgagaaaacc gatgtcatta aactggtgtc
     2281 tctggacctg aggtttgcac tcacatttcc atctggcggc cccataagca atctgtccta
     2341 cagataactc gtcctacaca aaacttagtc tcttttcagc tcagctctct cactctcaat
     2401 tatatctcct tacttccata tggcactgtt gtacactcat ttactcagag ccagaaacgt
     2461 cagcgtcatc ttggattttt cttatgctct ttctctctct agtcatatgc cagactttaa
     2521 actctgcttg aaagctttct cataagctct ttccttttcc ctttctactg ctttgcattt
     2581 gctacttaac ccttttcttc aggctgtttg ctttccagtc catcgttcgc tctgctgtta
     2641 ctcttctgcg tagtttctgt tacttgttgc tgaacatccc aatggcgctt tgaggaagat
     2701 gaactgagat gtgtcagggt gacttatttc catcatcgtc cttaggggag cttgggtagg
     2761 ggcaaggcgt gtagctggga cctaggtcca gacccctggc tctgccactg aacggctcag
     2821 gtgctttggg cagttactcc ccggcctcac tttgcacgtg tgcttaccta gtggagacaa
     2881 aagtacatac ctcggtagag cgcgcacgcc tgtaacccca ggttaactct ctaacctcga
     2941 gggggggccc ggtaccagct tttgttccct ttagtgaggg ttaatttcga gcttggcgta
     3001 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     3061 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     3121 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3181 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     3241 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3301 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3361 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3421 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3481 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3541 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3601 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3661 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3721 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     3781 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     3841 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     3901 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     3961 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4021 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4081 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4141 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4201 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4261 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4321 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4381 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4441 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4501 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4561 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4621 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4681 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4741 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     4801 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     4861 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     4921 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     4981 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5041 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5101 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa
     5161 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     5221 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     5281 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     5341 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     5401 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     5461 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     5521 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     5581 cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac
     5641 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
     5701 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
     5761 acgacggcca gtgaattgta atacgactca ctatagggcg aattggagct ccaccgcggt
     5821 ggcggccgct ctagaactag tggat
//
