LOCUS       sequence-364203-        5262 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PhCMV-driven triple nuclear localization 
            signal expression (PhCMV-3xNLS-pA). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5262)
  AUTHORS   Haellman V, Strittmatter T, Bertschi A, Stucheli P, Fussenegger M
  TITLE     A versatile plasmid architecture for mammalian synthetic biology 
            (VAMSyB).
  JOURNAL   Metab Eng. 2021 Jul;66:41-50. doi: 10.1016/j.ymben.2021.04.003. Epub
            2021 Apr 18.
  PUBMED    33857582
REFERENCE   2  (bases 1 to 5262)
  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: "Metab 
            Eng."; date: "2021-07"; volume: "66:41-50. doi"; pages: " 
            10.1016/j.ymben.2021.04.003. Epub 2021 Apr 18"
COMMENT     Sequence Label: pVH9-Tier1-PhCMV-3xNLS
FEATURES             Location/Qualifiers
     source          1..5262
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        92..471
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        472..675
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     626..646
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     720..739
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        720..738
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             782..802
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             806..826
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             830..850
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(905..922)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    911..1135
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1181..1609
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1268..1287)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1478..1499
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1618..1638)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1623..1952
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1803..1938
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1865..1884
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2019..2813
                     /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(2073..2092)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2683..2702
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2987..3108
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3024..3043)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3078..3097
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3157..3173)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3157..3173)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3170..3192)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3181..3197
                     /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(3205..3211,3212..3229,3230..3235))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3250..3271
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3388..3405)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3559..4147)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3639..3658)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4318..5109,5110..5178))
                     /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     4941..4960
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(5179..5262,1..21))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
       61 gggagatctc cacgcgtggt accctcgagt tgacattgat tattgagtag ttattaatag
      121 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
      181 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
      241 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
      301 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
      361 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
      421 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
      481 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
      541 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
      601 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
      661 tatataagca gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt
      721 aatacgactc actataggga gacccaagct gttcgaagcg gaattcacca tgactagtga
      781 tccaaaaaag aagagaaagg tagatccaaa aaagaagaga aaggtagatc caaaaaagaa
      841 gagaaaggta gctagcggat ccaccggtgt ctagaaagct ttgaggccgg cctgcaggga
      901 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
      961 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     1021 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     1081 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     1141 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     1201 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     1261 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     1321 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     1381 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     1441 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     1501 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     1561 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
     1621 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     1681 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     1741 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     1801 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     1861 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     1921 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     1981 tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     2041 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     2101 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     2161 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     2221 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     2281 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     2341 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     2401 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     2461 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     2521 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     2581 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     2641 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     2701 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     2761 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     2821 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     2881 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     2941 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     3001 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     3061 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     3121 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     3181 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     3241 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     3301 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3361 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     3421 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3481 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3541 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3601 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3661 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3721 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3781 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3841 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     3901 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     3961 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     4021 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4081 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     4141 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4201 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4261 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4321 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4381 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4441 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4501 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4561 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4621 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4681 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     4741 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     4801 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     4861 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     4921 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     4981 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     5041 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     5101 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     5161 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     5221 ttgtctcatg agcggataca tatttgaatg tatttagaaa aa
//
