LOCUS       sequence-340978-        3843 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPIE donor B4 (Zhong et al, eLife 2021), mNeonGreen translational
            phase (0-0), excised by ACTB i4 sgRNA or DRS-1 sgRNA (with SpCas9)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3843)
  AUTHORS   Zhong H, Ceballos CC, Massengill CI, Muniak MA, Ma L, Qin M, Petrie 
            SK, Mao T
  TITLE     High-fidelity, efficient, and reversible labeling of endogenous 
            proteins using CRISPR-based designer exon insertion.
  JOURNAL   Elife. 2021 Jun 8;10. pii: 64911. doi: 10.7554/eLife.64911.
  PUBMED    34100715
REFERENCE   2  (bases 1 to 3843)
  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"; journalName: "Elife."; 
            date: "2021-06-8"; volume: "10. pii: 64911. doi"; pages: " 
            10.7554/eLife.64911"
COMMENT     Sequence Label: pUC57-Amp_CRISPIE donor B4
FEATURES             Location/Qualifiers
     source          1..3843
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(31..48)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(202..790)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(282..301)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(961..1752,1753..1821))
                     /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     1584..1603
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1822..1926)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1994..2012
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2050..2072)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2172..2191
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2385..2407
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2399..2416
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2400..2416
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      2639..2648
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3643..3659)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3643..3659)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3656..3678)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3667..3683
                     /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(3691..3697,3698..3715,3716..3721))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3736..3757
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
       61 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
      121 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
      181 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
      241 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
      301 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
      361 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
      421 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
      481 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
      541 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
      601 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
      661 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
      721 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
      781 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
      841 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
      901 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
      961 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     1021 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     1081 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     1141 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     1201 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     1261 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     1321 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     1381 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     1441 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     1501 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     1561 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     1621 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     1681 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     1741 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     1801 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     1861 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     1921 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
     1981 attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga
     2041 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     2101 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg
     2161 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat
     2221 accgcacaga tgcgtaagga gaaaataccg catcaggcgc cattcgccat tcaggctgcg
     2281 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
     2341 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
     2401 taaaacgacg gccagtgaat tgacgcgtat tgggatacta gtaaaaggca aacactggtc
     2461 ggaggaattc gacgcttccg agtacggtac aggcggccgc gacgcttccg agtacggtac
     2521 aggcaaattg ctccagaagg gtgagaaggc tagagatgtg ggagaactgg gtggaaggca
     2581 gaatcgacca cgtctaaggt tgtgggttta ctgtgtctta cagttgaagg aaaccggtgc
     2641 caccatggtc agcaaaggcg aagaagacaa catggcatca ctgccagcga cacacgaact
     2701 tcatattttc ggctccatca atggtgtgga cttcgatatg gttggacagg gtactgggaa
     2761 ccctaacgat ggatatgagg agttgaactt gaagagtacc aagggcgatc ttcagttttc
     2821 accttggatt ctcgtacctc atataggtta tggctttcat caatatctgc cctatcctga
     2881 cggtatgtca cctttccaag cagctatggt tgacgggagc ggttatcagg ttcaccggac
     2941 aatgcagttt gaagatggag cctctttgac cgtaaattat cggtatactt atgagggcag
     3001 ccatattaaa ggcgaggctc aagttaaggg gactgggttc cccgcggatg gtcccgtaat
     3061 gacaaacagt ctgacggctg cagactggtg ccgctctaag aagacatatc caaatgacaa
     3121 gacgattatt agcactttca agtggtcata cactacagga aatggtaaac gctataggag
     3181 cactgcccgg accacgtata ctttcgcgaa gccgatggcg gcaaattatt tgaaaaacca
     3241 accgatgtat gtgttcagaa agacagaact caagcattct aaaactgaac tgaacttcaa
     3301 agagtggcag aaagccttta ctgatgtaat gggcatggac gaactgtaca agtccggagg
     3361 tagtgctggt ggtaccggac tcagatctgg cagcggtgga ggcagcgcag gtcctgccgc
     3421 agtaaggatt ctttcttcct gaatctctgg ggtgcattaa gggtattgcc ttagtagagg
     3481 ggcatgcccc aggggagtgg aggtggggtg agggtggagg acctgtaccg tactcggaag
     3541 cgtcgatatc cctgtaccgt actcggaagc gtcctgcaga aaaggcaaac actggtcgga
     3601 ggaagcttat cccaatggcg cgccgagctt ggcgtaatca tggtcatagc tgtttcctgt
     3661 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     3721 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     3781 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3841 agg
//
