LOCUS       sequence-218833-        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"
     primer_bind     8..24
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8..24
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             join(1016..1018,1019..1021,1022..1732)
                     /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(1061..1082)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1322..1341)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1669..1690
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2991..3009)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3030..3046)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3030..3046)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3043..3065)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3054..3070
                     /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(3078..3084,3085..3102,3103..3108))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3123..3144
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3261..3278)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3432..4020)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3512..3531)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4191..4982,4983..5051))
                     /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     4814..4833
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5052..5156)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5182..5637)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5319..5340)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5531..5550
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5764..5786
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5778..5795
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5779..5795
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5802..5821
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5802..5820
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 cggccgctct agaactagtg gatccgggcc cacactaaag ttagagaacc acaggctcgc
       61 tcacaaccct gacttctcca tgtcagttcc gatctttgcg aaccgcagac agggaaggtc
      121 ttctctcagg ggtcatgccc gcggccgccc tccacggcga ggtccgcact cgcgcagccg
      181 gccccgcggc cgcctcacct ggtcgcacac taccacgtcg aactcctcgt cggcgaggaa
      241 cagcacgtag agcgccagga aaaccatgcg cacgtaggcg cagacggcgg cgccgcggcc
      301 gccccagccc aggcctcgcg gcagccagtc cccggcacag cgcaccggta gctcgcggct
      361 ctcggcgaaa cagtggcccg ggtcgtagtg cgctgtccag atcttcacgc tacacccgcg
      421 cgcctgcagc gccagcgccg cgtccaacac cagccgctca gcgccgccca cgcccaggtc
      481 tgggtggagg aacagcaccg acggcttggg aaccgagtcc cgttcccggc cctgctcctc
      541 cgccatggcc ctggagccgc aactgcaccc cgcaccctga tgggggtctt ctgcgcaagc
      601 tccgcgctcg tagctcccag ctggccactg cgggccgacc ccgccctgcc gtacgtgcgt
      661 cagttaggcc acatcagcgc aaatctgtga gggtctagta actgcctgag aaaatatctt
      721 gtctgacccc ggttatattt ttccttcggt agggattgga ctttctgaag gacgttgtga
      781 tccaaaggaa ggaggccgga ggtctctact tcccatacag caggtaacta agttgtctgt
      841 agcagactgt ctacaggcat atcgtgagac gacccaggcg tccctggggt cagagaggac
      901 cttgcctgca agtccggggg cggggcctga gtcagtctcg ccagctgccg gtctttcggg
      961 ggctccgtaa ctttctatcc gtccgcgtca gcgccttgcc acactcatct ccaatatggt
     1021 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1081 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     1141 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1201 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     1261 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     1321 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     1381 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1441 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     1501 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     1561 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     1621 gagcacccag tccaagctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     1681 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agagcggcct
     1741 gagaagcatg gtatggcggc ccttccatga tccccgcctc tcccagaagc cctgactcct
     1801 cctgctttgc gccgtgcttt tcctctgtag ctcccttgct tcccccagcc tcgggtgtgg
     1861 gtgtctaggc cggggttctg gggcaggcct gccgcgctca cccgtctgtc tgcttgtctc
     1921 cctctacagc ctggtccgac ccccagtggc actaacgtgg gatcctcagg gcgctctccc
     1981 agcaaagcag tggccgcccg ggcggcggga tccactgtcc ggcagaggta aggaaccctg
     2041 cagttcgttc gcttccagac tcggagatag gacccagaac ctcgctgatt ctggggtgga
     2101 gaccctagca tgtgaagatt gacaaaggca aaatgagctt ctagtgacgt ggccgtggga
     2161 gtagttaaag gccttttggg aggaaggcga catttttttt ctcgttgctc agtttagggc
     2221 actactctta aaaaaggaaa gttaacaaac tggaatagag tcagagataa ctttgagaaa
     2281 accgatgtca ttaaactggt gtctctggac ctgaggtttg cactcacatt tccatctggc
     2341 ggccccataa gcaatctgtc ctacagataa ctcgtcctac acaaaactta gtctcttttc
     2401 agctcagctc tctcactctc aattatatct ccttacttcc atatggcact gttgtacact
     2461 catttactca gagccagaaa cgtcagcgtc atcttggatt tttcttatgc tctttctctc
     2521 tctagtcata tgccagactt taaactctgc ttgaaagctt tctcataagc tctttccttt
     2581 tccctttcta ctgctttgca tttgctactt aacccttttc ttcaggctgt ttgctttcca
     2641 gtccatcgtt cgctctgctg ttactcttct gcgtagtttc tgttacttgt tgctgaacat
     2701 cccaatggcg ctttgaggaa gatgaactga gatgtgtcag ggtgacttat ttccatcatc
     2761 gtccttaggg gagcttgggt aggggcaagg cgtgtagctg ggacctaggt ccagacccct
     2821 ggctctgcca ctgaacggct caggtgcttt gggcagttac tccccggcct cactttgcac
     2881 gtgtgcttac ctagtggaga caaaagtaca tacctcggta gagcgcgcac gcctgtaacc
     2941 ccaggttaac tctctaacct cgaggggggg cccggtacca gcttttgttc cctttagtga
     3001 gggttaattt cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     3061 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     3121 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3181 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3241 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3301 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3361 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3421 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3481 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3541 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3601 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3661 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3721 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     3781 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3841 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     3901 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     3961 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4021 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4081 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4141 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     4201 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     4261 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     4321 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     4381 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     4441 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     4501 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     4561 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     4621 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     4681 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     4741 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     4801 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     4861 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     4921 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     4981 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     5041 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     5101 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5161 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     5221 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     5281 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     5341 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     5401 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     5461 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     5521 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     5581 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
     5641 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     5701 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     5761 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac tcactatagg
     5821 gcgaattgga gctccaccgc ggtgg
//
