LOCUS       sequence-393127-        4789 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Pseudoalteromonas 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 4789)
  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 4789)
  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: pSL3617 (pCRISPR(BbsI)_Pse)
FEATURES             Location/Qualifiers
     source          1..4789
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(129..233)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(308..327)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        496..744
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     496..516
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     667..686
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     746..765
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        746..764
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    783..797
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     rep_origin      920..1348
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1007..1026)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1217..1238
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1357..1377)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1362..1691
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1542..1677
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1604..1623
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1758..2552
                     /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(1812..1831)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2422..2441
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2726..2847
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2763..2782)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2817..2836
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2896..2912)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2896..2912)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2909..2931)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2920..2936
                     /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(2944..2950,2951..2968,2969..2974))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2989..3010
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3127..3144)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3298..3886)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3378..3397)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4057..4789,1..59,60..128))
                     /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     4680..4699
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
       61 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
      121 atactcatac tcttcctttt tcattattat tgaagcattt atcagggtta ttgtctcatg
      181 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
      241 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
      301 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
      361 tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
      421 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
      481 gggccagata tacgcgaggg cctatttccc atgattcctt catatttgca tatacgatac
      541 aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa
      601 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
      661 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
      721 atcttgtgga aaggacgaaa caccgtaata cgactcacta tagggtctag aattcctgtg
      781 acctgccgta taggcagctg aagatcagtc ttcgatggtc gaagacgata attctgccga
      841 aaaggcagtg agtagtgtag cggccgcttt ttttcttctg aggcggaaag aaccagctgg
      901 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
      961 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     1021 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     1081 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     1141 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     1201 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     1261 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     1321 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
     1381 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     1441 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     1501 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     1561 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     1621 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     1681 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag
     1741 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     1801 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     1861 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     1921 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     1981 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     2041 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     2101 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     2161 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     2221 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     2281 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     2341 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     2401 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     2461 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     2521 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     2581 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     2641 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     2701 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     2761 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     2821 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     2881 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     2941 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     3001 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     3061 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     3121 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     3181 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     3241 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3301 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3361 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3421 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3481 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3541 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3601 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3661 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3721 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3781 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     3841 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3901 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3961 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4021 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     4081 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4141 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     4201 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     4261 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     4321 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     4381 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     4441 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     4501 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     4561 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     4621 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     4681 taataccgcg ccacatagca taactttaaa agtgctcatc attggaaaac gttcttcggg
     4741 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaa
//
