LOCUS       sequence-393053-        6658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human codon-optimized V. cholerae TnsC-VP64 from a CMV 
            promoter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6658)
  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 6658)
  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: pSL2783 (pTnsC-VP64_Vch)
FEATURES             Location/Qualifiers
     source          1..6658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(259..363)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(438..457)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        629..1008
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1009..1212
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1163..1183
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1257..1276
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1257..1275
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1323..1332
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2364..2384
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2430..2579
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     promoter        complement(2605..2623)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2606..2623)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2643..2660)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2649..2873
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2919..3347
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3006..3025)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3216..3237
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3356..3376)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3361..3690
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3541..3676
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3603..3622
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3757..4551
                     /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(3811..3830)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4421..4440
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4725..4846
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4762..4781)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4816..4835
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4895..4911)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4895..4911)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4908..4930)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4919..4935
                     /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(4943..4949,4950..4967,4968..4973))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4988..5009
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5126..5143)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5297..5885)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5377..5396)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6056..6658,1..189,190..258))
                     /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"
ORIGIN
        1 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
       61 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
      121 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
      181 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
      241 gaaatgttga atactcatac tcttcctttt tcattattat tgaagcattt atcagggtta
      301 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
      361 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
      421 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
      481 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
      541 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
      601 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
      661 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
      721 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      781 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      841 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      901 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      961 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
     1021 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     1081 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     1141 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     1201 ataagcagag ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat
     1261 acgactcact atagggagac ccaagcttgg taccgagctc ggatccctac ttccaatcca
     1321 atgccaccat gggctccgag acaagagagg caaggatctc tcgcgcaaag agggcctttg
     1381 tgagcacacc ctccgtgcgc aagatcctgt cctatatgga tcgctgccgg gacctgtccg
     1441 atctggagtc tgagcctacc tgtatgatgg tgtacggcgc ctctggcgtg ggcaagacca
     1501 cagtgatcaa gaagtatctg aatcagaata ggagggagag cgaggcagga ggcgacatca
     1561 tccccgtgct gcacatcgag ctgcctgaca acgccaagcc agtggatgcc gccagagagc
     1621 tgctggtgga gatgggcgac cctctggccc tgtacgagac agatctggcc agactgacaa
     1681 agaggctgac cgagctgatc ccagcagtgg gagtgaagct gatcatcatc gatgagttcc
     1741 agcacctggt ggaggagaga agcaacaggg tgctgacaca agtgggcaac tggctgaaga
     1801 tgatcctgaa taagaccaag tgccctatcg tgatctttgg catgccatat agcaaggtgg
     1861 tgctccaggc caattctcag ctgcacggca gattcagcat ccaggtggag ctgaggccat
     1921 tttcctacca gggaggaagg ggcgtgttca agacctttct ggagtatctg gacaaggccc
     1981 tgcccttcga gaagcaggca ggactggcca acgagtctct ccagaagaag ctgtacgcct
     2041 tttcccaggg caacatgcgc tctctgcgga atctgatcta tcaggcctcc atcgaggcca
     2101 tcgacaatca gcacgaaacc atcacagagg aggatttcgt gtttgccagc aagctgacat
     2161 ccggcgacaa gccaaacagc tggaagaatc ccttcgagga gggcgtggag gtgacagagg
     2221 atatgctgcg gccacccccc aaggacatcg gctgggagga ttacctgaga cactccaccc
     2281 ccagggtgtc taagcctggc agaaacaaga atttctttga gggctccgga aaacggacag
     2341 ccgacggaag cgagttcgag tcaccgaaaa agaagcgaaa agttggtggt agtggtggaa
     2401 gcggcgaggc cagcggttcc ggacgggctg acgcattgga cgattttgat ctggatatgc
     2461 tgggaagtga cgccctcgat gattttgacc ttgacatgct tggttcggat gcccttgatg
     2521 actttgacct cgacatgctc ggcagtgacg cccttgatga tttcgacctg gacatgctga
     2581 ttaactctag ataaagggcc ctattctata gtgtcaccta aatgctagag ctcgctgatc
     2641 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2701 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2761 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2821 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2881 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     2941 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3001 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3061 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3121 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3181 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3241 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     3301 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     3361 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3421 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     3481 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3541 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3601 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     3661 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     3721 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     3781 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3841 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     3901 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     3961 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     4021 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     4081 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     4141 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     4201 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     4261 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     4321 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     4381 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     4441 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     4501 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     4561 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     4621 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4681 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4741 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4801 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4861 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4921 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4981 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5041 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5101 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5161 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5221 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5281 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5341 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5401 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5461 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5521 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5581 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5641 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5701 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5761 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5821 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5881 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5941 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6001 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6061 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6121 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6181 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6241 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6301 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6361 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6421 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6481 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6541 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6601 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctc
//
