LOCUS       sequence-398288-        6528 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human codon-optimized V. cholerae Cas7 from a CMV 
            promoter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6528)
  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 6528)
  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: pSL2622 (pCas7_Vch)
FEATURES             Location/Qualifiers
     source          1..6528
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(124..228)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(303..322)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        494..873
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        874..1077
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1028..1048
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1122..1141
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1122..1140
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1172..1181
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1217..1237
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(2340..2358)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2341..2358)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2378..2395)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2384..2608
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2654..3082
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2741..2760)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2951..2972
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3091..3111)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3096..3425
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3276..3411
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3338..3357
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3492..4286
                     /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(3546..3565)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4156..4175
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4460..4581
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4497..4516)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4551..4570
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4630..4646)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4630..4646)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4643..4665)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4654..4670
                     /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(4678..4684,4685..4702,4703..4708))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4723..4744
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4861..4878)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5032..5620)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5112..5131)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5791..6528,1..54,55..123))
                     /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     6414..6433
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
       61 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
      121 catactcttc ctttttcatt attattgaag catttatcag ggttattgtc tcatgagcgg
      181 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
      241 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc
      301 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg
      361 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
      421 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
      481 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
      541 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      601 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      661 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      721 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      781 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      841 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      901 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      961 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     1021 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc
     1081 tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg
     1141 gagacccaag cttggtaccg agctcggatc cgccaccatg ggcaaacgga cagccgacgg
     1201 aagcgagttc gagtcaccga aaaagaagcg aaaagttggc tccggaatga agctgccaac
     1261 caacctggcc tacgagcgga gcatcgaccc cagcgacgtg tgcttcttcg tggtgtggcc
     1321 agacgatagg aaaacccccc tgacatacaa ttcccgcaca ctgctgggac agatggaggc
     1381 agccagcctg gcatacgacg tgtccggcca gcctatcaag tctgccaccg cagaggccct
     1441 ggcacagggc aaccctcacc aggtggattt ctgccatgtg ccatacggcg ccagccacat
     1501 cgagtgtagc ttctccgtgt cttttagctc cgagctgcgg cagccatata agtgtaattc
     1561 tagcaaggtg aagcagacac tggtgcagct ggtggagctg tacgaaacca agatcggctg
     1621 gacagagctg gccacccggt atctgatgaa tatctgcaac ggcaagtggc tgtggaagaa
     1681 tacaagaaag gcctactgtt ggaacatcgt gctgaccccc tggccttgga atggcgagaa
     1741 agtgggcttc gaggacatca ggaccaacta tacatcccgc caggacttca agaacaataa
     1801 gaattggtct gccatcgtgg agatgatcaa gacagccttc tcctctaccg acggcctggc
     1861 catctttgag gtgagggcca cactgcacct gccaaccaac gcaatggtgc gccctagcca
     1921 ggtgttcaca gagaaggaga gcggctccaa gtctaagagc aagacccaga actctagggt
     1981 gttccagagc accacaatcg atggagagcg gagcccaatc ctgggagcct ttaagacagg
     2041 cgccgccatc gccaccatcg acgattggta tccagaggca accgagcctc tgagggtggg
     2101 ccgctttgga gtgcacaggg aggacgtgac atgctacaga cacccatcta ccggcaagga
     2161 tttctttagc atcctccagc aggccgagca ctatatcgag gtgctgagtg ccaacaagac
     2221 acccgcccag gaaaccatca atgacatgca cttcctgatg gccaacctga tcaagggcgg
     2281 catgtttcag cacaagggcg actaattaca ttggaagtgg ataatctaga gggccctatt
     2341 ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc
     2401 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     2461 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     2521 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     2581 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     2641 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2701 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2761 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2821 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     2881 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     2941 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3001 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3061 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3121 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3181 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3241 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3301 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3361 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3421 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     3481 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     3541 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     3601 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     3661 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     3721 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     3781 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     3841 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     3901 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     3961 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     4021 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     4081 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4141 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     4201 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4261 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     4321 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     4381 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     4441 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     4501 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4561 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     4621 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4681 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4741 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4801 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4861 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4921 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4981 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5041 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5101 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5161 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5221 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5281 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5341 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5401 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5461 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5521 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5581 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5641 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5701 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5761 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5821 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5881 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     5941 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6001 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6061 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6121 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6181 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6241 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6301 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6361 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6421 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6481 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccac
//
