LOCUS       sequence-393167-        6726 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human codon-optimized E. coli ClpX from a CMV promoter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6726)
  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 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: "Nat 
            Biotechnol. 2023 Mar 29. doi: 10.1038/s41587-023-01748-1."
COMMENT     Sequence Label: pSL4393 (pClpX_Eco)
FEATURES             Location/Qualifiers
     source          1..6726
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             24..44
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1361..1378)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1367..1591
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1637..2065
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1724..1743)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1934..1955
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2074..2094)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2079..2408
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2259..2394
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2321..2340
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2475..3269
                     /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(2529..2548)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3139..3158
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3443..3564
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3480..3499)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3534..3553
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3613..3629)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3613..3629)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3626..3648)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3637..3653
                     /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(3661..3667,3668..3685,3686..3691))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3706..3727
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3844..3861)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4015..4603)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4095..4114)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4774..5565,5566..5634))
                     /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     5397..5416
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5635..5739)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5814..5833)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6005..6384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6385..6588
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6539..6559
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6633..6652
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6633..6651
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      6705..6714
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 ccgacggaag cgagttcgag tcaccgaaaa agaagcgaaa agttggctcc ggaatgaccg
       61 acaagcggaa ggacggctct ggcaagctgc tgtactgcag cttctgcggc aaatctcagc
      121 acgaggtgcg gaagctgatc gccggaccta gcgtgtatat ctgtgatgag tgcgtggatc
      181 tgtgcaacga catcatccgg gaagaaatca aggaagtggc ccctcacagg gagcggagcg
      241 ccctgcctac accacatgag atcagaaatc acctggatga ttacgtgatc ggccaggagc
      301 aggctaaaaa ggtcctggcc gtggccgtgt acaaccacta caagagactg cgcaacggcg
      361 acaccagcaa tggcgtggag ctgggcaaga gcaacatcct gctgattggc cctaccggaa
      421 gcggaaagac actgctggct gaaaccctgg ccagactgct ggacgtgcct ttcacaatgg
      481 ccgacgccac aacactgacc gaggctggct acgtgggcga ggacgtggag aacatcatcc
      541 agaagctgct tcagaagtgt gactacgacg tgcagaaagc ccagagaggc atcgtgtaca
      601 tcgacgagat cgataagatc agcagaaagt ccgacaaccc cagcatcacc cgggacgtga
      661 gcggcgaggg cgttcagcag gccctgctga agctgatcga ggggaccgtg gccgctgtgc
      721 ccccccaggg cggaagaaag caccctcagc aagagttcct gcaagtggac accagcaaga
      781 tcctgttcat ctgcggcggc gccttcgccg gcctggacaa agtgatctcc caccgggtgg
      841 agacaggatc tggaatcggc ttcggcgcca ccgtgaaggc caagtctgat aaagccagcg
      901 agggcgaact gctcgctcaa gtggaacccg aggacctgat caagttcggc ctgatccctg
      961 agttcatcgg cagactgcca gtggtggcta cactcaacga gctgagcgag gaagcactga
     1021 ttcagatcct gaaagagcct aagaacgccc tgaccaagca gtaccaggcc ctgtttaacc
     1081 tggaaggcgt ggacctggaa tttagagatg aggccctcga cgccatcgcc aagaaggcta
     1141 tggccagaaa gaccggcgct cggggcctga gaagcatcgt ggaagccgcc ctgctggata
     1201 caatgtacga cctgcccagc atggaagatg tggaaaaggt cgtgatcgac gagagcgtta
     1261 tcgacggcca gagcaagcct ctgctgatct acggcaaacc tgaagcccag caggcttctg
     1321 gcgagtgaat ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt
     1381 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     1441 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     1501 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     1561 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     1621 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     1681 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     1741 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     1801 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     1861 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     1921 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     1981 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     2041 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     2101 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     2161 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     2221 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     2281 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     2341 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     2401 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat
     2461 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     2521 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     2581 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     2641 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     2701 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     2761 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     2821 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     2881 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     2941 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     3001 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     3061 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     3121 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     3181 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     3241 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     3301 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     3361 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     3421 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     3481 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     3541 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
     3601 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     3661 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     3721 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3781 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3841 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3901 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3961 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     4021 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     4081 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     4141 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     4201 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     4261 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     4321 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     4381 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     4441 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4501 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4561 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4621 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4681 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4741 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     4801 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     4861 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     4921 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     4981 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     5041 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     5101 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     5161 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     5221 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     5281 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     5341 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     5401 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     5461 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     5521 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     5581 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     5641 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     5701 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5761 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
     5821 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
     5881 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
     5941 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
     6001 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
     6061 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     6121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     6181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     6241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     6301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     6361 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
     6421 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     6481 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     6541 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
     6601 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
     6661 gctggctagc gtttaaactt aagcttggta ccgagctcgg atccgccacc atgggcaaac
     6721 ggacag
//
