LOCUS       sequence-434398-        4215 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of type III-A crRNA from Streptococcus 
            thermophilus within 5`UTR of mCherry transcript.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4215)
  AUTHORS   Nemudraia A, Nemudryi A, Wiedenheft B
  TITLE     Repair of CRISPR-guided RNA breaks enables site-specific RNA 
            excision in human cells.
  JOURNAL   Science. 2024 Apr 25:eadk5518. doi: 10.1126/science.adk5518.
  PUBMED    38662916
REFERENCE   2  (bases 1 to 4215)
  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: "Science. 
            2024 Apr 25:eadk5518. doi: 10.1126/science.adk5518."
COMMENT     Sequence Label: pAN053
FEATURES             Location/Qualifiers
     source          1..4215
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      14..602
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     503..522
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     756..773
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    890..911
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(926..931,932..949,950..956)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    964..980
                     /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)."
     primer_bind     969..991
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     988..1004
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     988..1004
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1075..1299
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1288..1305
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(1328..1345)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1355..1381)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(1391..2098)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1666..1685)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1935..1953
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      2095..2104
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2240..2443)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2269..2289)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2444..2823
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2995..3014
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3093..3197
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3198..3266,3267..4058)
                     /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     complement(3416..3435)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
       61 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
      121 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
      181 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
      241 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
      301 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
      361 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
      421 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
      481 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
      541 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
      601 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
      661 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
      721 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
      781 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
      841 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
      901 agctcactca ttaggcaccc taggctttac actttatgct tccggctcgt atgttgtgtg
      961 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
     1021 tctagctaga ggtcgacggt atcgagcccc agctggttct ttccgcctca gaagccatag
     1081 agcccaccgc atccccagca tgcctgctat tgtcttccca atcctccccc ttgctgtcct
     1141 gccccacccc accccccaga atagaatgac acctactcag acaatgcgat gcaattttct
     1201 cattttatta ggaaaggaca gtgggagtgg caccttccag ggtcaaggaa ggcacggggg
     1261 aggggcaaac aacagatggc tggcaactag aaggcacagt cgaggctgat cagcgggttt
     1321 aaactcaatg gtgatggtga tgatgaccgg tttaagcgta atctggaaca tcgtatgggt
     1381 atccggatcc cttgtacagc tcgtccatgc cgccggtgga gtggcggccc tcggcgcgtt
     1441 cgtactgttc cacgatggtg tagtcctcgt tgtgggaggt gatgtccaac ttgatgttga
     1501 cgttgtaggc gccgggcagc tgcacgggct tcttggcctt gtaggtggtc ttgacctcag
     1561 cgtcgtagtg gccgccgtcc ttcagcttca gcctctgctt gatctcgccc ttcagggcgc
     1621 cgtcctcggg gtacatccgc tcggaggagg cctcccagcc catggtcttc ttctgcatta
     1681 cggggccgtc ggaggggaag ttggtgccgc gcagcttcac cttgtagatg aactcgccgt
     1741 cctgcaggga ggagtcctgg gtcacggtca ccacgccgcc gtcctcgaag ttcatcacgc
     1801 gctcccactt gaagccctcg gggaaggaca gcttcaagta gtcggggatg tcggcggggt
     1861 gcttcacgta ggccttggag ccgtacatga actgagggga caggatgtcc caggcgaagg
     1921 gcagggggcc acccttggtc accttcagct tggcggtctg ggtgccctcg taggggcggc
     1981 cctcgccctc gccctcgatc tcgaactcgt ggccgttcac ggagccctcc atgtgcacct
     2041 tgaagcgcat gaactccttg atgatggcca tgttatcctc ctcgcccttg ctcaccatgg
     2101 tggcggccgc gtcccctctc gaggtaatta ggtttatatc agagacgctg aatcgcttca
     2161 tcgcgtctcg gtttccgtcc cctctcgagg taattaggtt tatatcggat ctctagcgga
     2221 tctgacggtt cactaaacca gctctgctta tatagacctc ccaccgtaca cgcctaccgc
     2281 ccatttgcgt caatggggcg gagttgttac gacattttgg aaagtcccgt tgattttggt
     2341 gccaaaacaa actcccattg acgtcaatgg ggtggagact tggaaatccc cgtgagtcaa
     2401 accgctatcc acgcccattg atgtactgcc aaaaccgcat caccatggta atagcgatga
     2461 ctaatacgta gatgtactgc caagtaggaa agtcccataa ggtcatgtac tgggcataat
     2521 gccaggcggg ccatttaccg tcattgacgt caataggggg cgtacttggc atatgataca
     2581 cttgatgtac tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga
     2641 aagtccctat tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt
     2701 cgttgggcgg tcagccaggc gggccattta ccgtaagtta tgtaacgcgg aactccatat
     2761 atgggctatg aactaatgac cccgtaattg attactatta ataactagtc aataatcaat
     2821 gtcaacgcgt atatctggcc cgtacatcgc gaagcagcgc aaaacgccta accctaagca
     2881 gattcttcat gcaattgtcg gtcaagcctt gccttgttgt agcttaaatt ttgctcgcgc
     2941 actactcagc gacctccaac acacaagcag ggagcagata ctggcttaac tatgcggcat
     3001 cagagcagat tgtactgaga gtagacccta ggggatcggg agatcgatct cccgatccgt
     3061 cgacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     3121 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3181 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     3241 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     3301 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     3361 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     3421 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     3481 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     3541 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     3601 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     3661 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     3721 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     3781 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     3841 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     3901 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     3961 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     4021 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     4081 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     4141 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagtgtcag
     4201 accccgtaga aaaga
//
