LOCUS       sequence-393701-        7392 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human codon-optimized V. cholerae Cas8 from a CMV 
            promoter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7392)
  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 7392)
  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: pSL2621 (pCas8_Vch)
FEATURES             Location/Qualifiers
     source          1..7392
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      913..922
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             958..978
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(2945..2963)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2946..2963)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2983..3000)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2989..3213
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3259..3687
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3346..3365)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3556..3577
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3696..3716)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3701..4030
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3881..4016
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3943..3962
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4097..4891
                     /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(4151..4170)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4761..4780
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5065..5186
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5102..5121)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5156..5175
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5235..5251)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5235..5251)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5248..5270)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5259..5275
                     /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(5283..5289,5290..5307,5308..5313))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5328..5349
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5466..5483)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5637..6225)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5717..5736)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6396..7187,7188..7256))
                     /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     7019..7038
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7257..7361)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat ccgccaccat gggcaaacgg acagccgacg gaagcgagtt cgagtcaccg
      961 aaaaagaagc gaaaagttgg ctccggaatg cagaccctga aggagctgat cgccagcaac
     1021 cccgacgatc tgaccacaga gctgaagagg gccttccgcc ccctgacacc tcacatcgcc
     1081 atcgacggca atgagctgga tgccctgacc atcctggtga acctgacaga caagaccgac
     1141 gatcagaagg acctgctgga tcgggccaag tgtaagcaga agctgagaga tgagaagtgg
     1201 tgggcctcct gcatcaactg cgtgaactac aggcagtctc acaacccaaa gttccccgac
     1261 atccgcagcg agggcgtgat cagaacacag gccctgggcg agctgcccag ctttctgctg
     1321 tctagctcca agatcccacc ctaccactgg tcttatagcc acgactctaa gtatgtgaat
     1381 aagagcgcct tcctgaccaa cgagttttgc tgggatggcg agatcagctg tctgggcgag
     1441 ctgctgaagg acgccgatca ccctctgtgg aacacactga agaagctggg ctgctcccag
     1501 aaaacctgta aggcaatggc caagcagctg gccgacatca cactgaccac aatcaatgtg
     1561 accctggccc ccaactacct gacacagatc tctctgcctg actccgatac atcttatatc
     1621 tccctgtctc ctgtggccag cctgtccatg cagagccact tccaccagag gctccaggat
     1681 gagaacaggc actccgccat cacccggttc agccggacca caaatatggg agtgacagca
     1741 atgacctgcg gaggagcctt caggatgctg aaaagcggcg ccaagttttc tagccctcca
     1801 caccaccggc tgaacagcaa gagatcctgg ctgacctccg agcatgtgca gtctctgaag
     1861 cagtaccagc ggctgaataa gagcctgatc ccagagaact ccagaatcgc cctgcggaga
     1921 aagtataaga tcgagctgca aaatatggtg cgctcttggt tcgccatgca ggaccacaca
     1981 ctggatagca atatcctgat ccagcacctg aaccacgacc tgtcctacct gggcgccacc
     2041 aagcggttcg cctatgatcc cgccatgaca aagctgttta ccgagctgct gaagagagag
     2101 ctgtctaaca gcatcaacaa tggcgagcag cacaccaatg gcagctttct ggtgctgcct
     2161 aacatcagag tgtgcggagc aaccgccctg tcctctcctg tgacagtggg catcccatcc
     2221 ctgaccgcct tctttggctt cgtgcacgcc tttgagagga atatcaaccg caccacaagc
     2281 tccttcaggg tggagagctt tgccatctgc gtgcaccagc tgcatgtgga gaagcgcggc
     2341 ctgacagccg agttcgtgga gaagggcgac ggaacaatct ccgccccagc aaccagggac
     2401 gattggcagt gtgatgtggt gttctctctg atcctgaata ccaactttgc ccagcacatc
     2461 gaccaggata cactcgtcac ctctctgcca aagaggctgg caaggggaag cgccaagatc
     2521 gccatcgacg atttcaagca catcaactcc ttttctacac tggaaaccgc aatcgagagc
     2581 ctgccaatcg aggcaggcag atggctgagt ctgtacgccc agtccaacaa taacctgtct
     2641 gacctgctgg ccgccatgac agaggatcac cagctgatgg ccagctgcgt gggctaccac
     2701 ctgctggagg agcctaaaga caagccaaac tccctcagag gctataagca cgccatcgcc
     2761 gagtgtatca tcggcctgat caattccatc accttctcta gcgagacaga tcccaacacc
     2821 atcttttgga gcctgaagaa ttaccagaac tatctggtgg tgcagcctcg ctccatcaat
     2881 gacgaaacca ccgataagtc ctctctgtaa ttacattgga agtggataat ctagagggcc
     2941 ctattctata gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta
     3001 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     3061 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     3121 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     3181 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     3241 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3301 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3361 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3421 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     3481 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3541 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3601 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     3661 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     3721 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3781 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3841 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     3901 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     3961 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     4021 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg
     4081 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     4141 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     4201 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     4261 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     4321 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     4381 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     4441 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     4501 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     4561 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     4621 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     4681 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     4741 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     4801 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     4861 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     4921 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     4981 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     5041 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     5101 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     5161 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     5221 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     5281 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     5341 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5401 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     5461 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     5521 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     5581 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     5641 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     5701 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     5761 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     5821 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5881 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5941 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     6001 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     6061 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     6121 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     6181 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     6241 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     6301 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     6361 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     6421 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     6481 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     6541 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     6601 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     6661 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     6721 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     6781 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     6841 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6901 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6961 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     7021 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     7081 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     7141 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     7201 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     7261 ttcctttttc attattattg aagcatttat cagggttatt gtctcatgag cggatacata
     7321 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     7381 ccacctgacg tc
//
