LOCUS       sequence-393087-        4792 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses V. cholerae CRISPR RNA from a U6 promoter. The CRISPR 
            array contains two BbsI sites for spacer cloning. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4792)
  AUTHORS   Lampe GD, King RT, Halpin-Healy TS, Klompe SE, Hogan MI, Vo PLH, 
            Tang S, Chavez A, Sternberg SH
  TITLE     Targeted DNA integration in human cells without double-strand breaks
            using CRISPR-associated transposases.
  JOURNAL   Nat Biotechnol. 2023 Mar 29. doi: 10.1038/s41587-023-01748-1.
  PUBMED    36991112
REFERENCE   2  (bases 1 to 4792)
  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: "Nat 
            Biotechnol. 2023 Mar 29. doi: 10.1038/s41587-023-01748-1."
COMMENT     Sequence Label: pSL0812 (pCRISPR(BbsI)_Vch)
FEATURES             Location/Qualifiers
     source          1..4792
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        55..303
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     55..75
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     226..245
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     305..324
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        305..323
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      482..910
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(569..588)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     779..800
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(919..939)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        924..1253
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1104..1239
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1166..1185
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1320..2114
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1374..1393)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1984..2003
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2288..2409
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2325..2344)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2379..2398
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2458..2474)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2458..2474)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2471..2493)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2482..2498
                     /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(2506..2512,2513..2530,2531..2536))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2551..2572
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2689..2706)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2860..3448)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2940..2959)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3619..4410,4411..4479))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVMLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4242..4261
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4480..4584)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4659..4678)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgagggc
       61 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
      121 ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
      181 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
      241 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac
      301 accgtaatac gactcactat agggtctaga attcctgtga actgccgagt aggtagctga
      361 taaccagtct tcgatggtcg aagacgagtg aactgccgag taggtagctg ataacgttgc
      421 agcggccgct ttttttcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
      481 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
      541 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
      601 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
      661 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
      721 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
      781 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
      841 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
      901 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
      961 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     1021 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     1081 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     1141 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     1201 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     1261 cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca
     1321 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     1381 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     1441 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     1501 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     1561 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     1621 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     1681 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     1741 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     1801 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     1861 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     1921 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     1981 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     2041 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     2101 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     2161 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     2221 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     2281 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     2341 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     2401 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     2461 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     2521 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     2581 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     2641 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     2701 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     2761 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     2821 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     2881 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     2941 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3001 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3061 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3121 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3181 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3241 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     3301 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     3361 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     3421 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     3481 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     3541 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     3601 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     3661 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     3721 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     3781 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     3841 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     3901 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     3961 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     4021 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     4081 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4141 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4201 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     4261 cataacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     4321 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     4381 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     4441 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcattatta
     4501 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     4561 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg
     4621 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
     4681 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
     4741 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tc
//
