LOCUS       sequence-467887-        6726 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human-codon optimized E. coli ClpX in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6726)
  AUTHORS   Witte IP, Lampe GD, Eitzinger S, Miller SM, Berrios KN, McElroy AN, 
            King RT, Stringham OG, Gelsinger DR, Vo PLH, Chen AT, Tolar J, 
            Osborn MJ, Sternberg SH, Liu DR
  TITLE     Programmable gene insertion in human cells with a laboratory-evolved
            CRISPR-associated transposase.
  JOURNAL   Science. 2025 May 15;388(6748):eadt5199. doi: 
            10.1126/science.adt5199. Epub 2025 May 15.
  PUBMED    40373119
REFERENCE   2  (bases 1 to 6726)
  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: "Science."; 
            date: "2025-05-15"; volume: "388(6748):eadt5199. doi"; pages: " 
            10.1126/science.adt5199. Epub 2025 May 15"
COMMENT     Sequence Label: pCMV-ClpX
FEATURES             Location/Qualifiers
     source          1..6726
                     /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      935..944
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             980..1000
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2317..2334)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2323..2547
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2593..3021
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2680..2699)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2890..2911
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3030..3050)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3035..3364
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3215..3350
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3277..3296
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3431..4225
                     /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(3485..3504)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4095..4114
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4399..4520
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4436..4455)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4490..4509
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4569..4585)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4569..4585)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4582..4604)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4593..4609
                     /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(4617..4623,4624..4641,4642..4647))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4662..4683
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4800..4817)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4971..5559)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5051..5070)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5730..6521,6522..6590))
                     /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     6353..6372
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6591..6695)
                     /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 gctggctagc
      901 gtttaaactt aagcttggta ccgagctcgg atccgccacc atgggcaaac ggacagccga
      961 cggaagcgag ttcgagtcac cgaaaaagaa gcgaaaagtt ggctccggaa tgaccgacaa
     1021 gcggaaggac ggctctggca agctgctgta ctgcagcttc tgcggcaaat ctcagcacga
     1081 ggtgcggaag ctgatcgccg gacctagcgt gtatatctgt gatgagtgcg tggatctgtg
     1141 caacgacatc atccgggaag aaatcaagga agtggcccct cacagggagc ggagcgccct
     1201 gcctacacca catgagatca gaaatcacct ggatgattac gtgatcggcc aggagcaggc
     1261 taaaaaggtc ctggccgtgg ccgtgtacaa ccactacaag agactgcgca acggcgacac
     1321 cagcaatggc gtggagctgg gcaagagcaa catcctgctg attggcccta ccggaagcgg
     1381 aaagacactg ctggctgaaa ccctggccag actgctggac gtgcctttca caatggccga
     1441 cgccacaaca ctgaccgagg ctggctacgt gggcgaggac gtggagaaca tcatccagaa
     1501 gctgcttcag aagtgtgact acgacgtgca gaaagcccag agaggcatcg tgtacatcga
     1561 cgagatcgat aagatcagca gaaagtccga caaccccagc atcacccggg acgtgagcgg
     1621 cgagggcgtt cagcaggccc tgctgaagct gatcgagggg accgtggccg ctgtgccccc
     1681 ccagggcgga agaaagcacc ctcagcaaga gttcctgcaa gtggacacca gcaagatcct
     1741 gttcatctgc ggcggcgcct tcgccggcct ggacaaagtg atctcccacc gggtggagac
     1801 aggatctgga atcggcttcg gcgccaccgt gaaggccaag tctgataaag ccagcgaggg
     1861 cgaactgctc gctcaagtgg aacccgagga cctgatcaag ttcggcctga tccctgagtt
     1921 catcggcaga ctgccagtgg tggctacact caacgagctg agcgaggaag cactgattca
     1981 gatcctgaaa gagcctaaga acgccctgac caagcagtac caggccctgt ttaacctgga
     2041 aggcgtggac ctggaattta gagatgaggc cctcgacgcc atcgccaaga aggctatggc
     2101 cagaaagacc ggcgctcggg gcctgagaag catcgtggaa gccgccctgc tggatacaat
     2161 gtacgacctg cccagcatgg aagatgtgga aaaggtcgtg atcgacgaga gcgttatcga
     2221 cggccagagc aagcctctgc tgatctacgg caaacctgaa gcccagcagg cttctggcga
     2281 gtgaatctag agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     2341 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     2401 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     2461 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     2521 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     2581 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     2641 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     2701 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     2761 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     2821 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     2881 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     2941 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     3001 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
     3061 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     3121 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3181 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     3241 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     3301 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     3361 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg
     3421 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     3481 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     3541 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     3601 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     3661 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     3721 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     3781 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     3841 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     3901 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     3961 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     4021 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     4081 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     4141 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     4201 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     4261 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     4321 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     4381 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4441 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     4501 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     4561 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     4621 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     4681 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4741 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     4801 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4861 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4921 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4981 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5041 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5101 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5161 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5221 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5281 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5341 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5401 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5461 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5521 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5581 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     5641 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     5701 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     5761 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     5821 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     5881 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     5941 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6001 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6061 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6121 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6181 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6241 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6301 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6361 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6421 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6481 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6541 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6601 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     6661 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     6721 gacgtc
//
