LOCUS       sequence-364613-        6198 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PhCMV-driven iRFP670 that is localized to the
            nucleus (PhCMV-3xNLS-iRFP670-pA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6198)
  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 6198)
  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: pTS1026-Tier1-PhCMV-3xNLS-iRFP670
FEATURES             Location/Qualifiers
     source          1..6198
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        68..447
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        448..651
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     602..622
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     696..715
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        696..714
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             758..778
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     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             833..1762
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 670 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP670
                     /note="derived from Rhodopseudomonas palustris RpBphP6"
                     /translation="ARKVDLTSCDREPIHIPGSIQPCGCLLACDAQAVRITRITENAGA
                     FFGRETPRVGELLADYFGETEAHALRNALAQSSDPKRPALIFGWRDGLTGRTFDISLHR
                     HDGTSIIEFEPAAAEQADNPLRLTRQIIARTKELKSLEEMAARVPRYLQAMLGYHRVML
                     YRFADDGSGMVIGEAKRSDLESFLGQHFPASLVPQQARLLYLKNAIRVVSDSRGISSRI
                     VPEHDASGAALDLSFAHLRSISPCHLEFLRNMGVSASMSLSIIIDGTLWGLIICHHYEP
                     RAVPMAQRVAAEMFADFLSLHFTAAHHQR"
     primer_bind     complement(1817..1834)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1823..2047
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2093..2521
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2180..2199)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2390..2411
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2530..2550)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2535..2864
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2715..2850
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2777..2796
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2931..3725
                     /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(2985..3004)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3595..3614
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3899..4020
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3936..3955)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3990..4009
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4069..4085)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4069..4085)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4082..4104)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4093..4109
                     /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(4117..4123,4124..4141,4142..4147))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4162..4183
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4300..4317)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4471..5059)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4551..4570)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5230..6021,6022..6090))
                     /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     5853..5872
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6091..6195)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctccacg cgtggtaccc
       61 tcgagttgac attgattatt gagtagttat taatagtaat caattacggg gtcattagtt
      121 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      181 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      241 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
      301 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      361 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      421 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
      481 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
      541 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
      601 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta
      661 actagagaac ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc
      721 caagctgttc gaagcggaat tcaccatgac tagtgatcca aaaaagaaga gaaaggtaga
      781 tccaaaaaag aagagaaagg tagatccaaa aaagaagaga aaggtagcta gtgcgcgtaa
      841 ggtcgatctc acctcctgcg atcgcgagcc gatccacatc cccggcagca ttcagccgtg
      901 cggctgcctg cttgcctgcg acgcgcaggc ggtgcggatc acgcgcatta cggaaaatgc
      961 cggcgcgttc tttggacgcg aaactccgcg ggtcggtgag ctactcgccg attacttcgg
     1021 cgagaccgaa gcccatgcgc tgcgcaacgc actggcgcag tcctccgatc caaagcgacc
     1081 ggcgctgatc ttcggttggc gcgacggcct gaccggccgc accttcgaca tctcactgca
     1141 tcgccatgac ggtacatcga tcatcgagtt cgagcctgcg gcggccgaac aggccgacaa
     1201 tccgctgcgg ctgacgcggc agatcatcgc gcgcaccaaa gaactgaagt cgctcgaaga
     1261 gatggccgca cgggtgccgc gctatctgca ggcgatgctc ggctatcacc gcgtgatgtt
     1321 gtaccgcttc gcggacgacg gctccgggat ggtgatcggc gaggcgaagc gcagcgacct
     1381 cgagagcttt ctcggtcagc actttccggc gtcgctggtc ccgcagcagg cgcggctact
     1441 gtacttgaag aacgcgatcc gcgtggtctc ggattcgcgc ggcatcagca gccggatcgt
     1501 gcccgagcac gacgcctccg gcgccgcgct cgatctgtcg ttcgcgcacc tgcgcagcat
     1561 ctcgccctgc catctcgaat ttctgcggaa catgggcgtc agcgcctcga tgtcgctgtc
     1621 gatcatcatt gacggcacgc tatggggatt gatcatctgt catcattacg agccgcgtgc
     1681 cgtgccgatg gcgcagcgcg tcgcggccga aatgttcgcc gacttcttat cgctgcactt
     1741 caccgccgcc caccaccaac gcgctagcgg atccaccggt gtctagaaag ctttgaggcc
     1801 ggcctgcagg gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     1861 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     1921 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     1981 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     2041 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     2101 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     2161 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     2221 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     2281 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     2341 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     2401 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     2461 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     2521 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     2581 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     2641 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     2701 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     2761 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     2821 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     2881 gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac
     2941 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     3001 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     3061 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     3121 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     3181 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     3241 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     3301 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     3361 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     3421 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     3481 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     3541 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     3601 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     3661 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     3721 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     3781 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     3841 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     3901 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     3961 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4021 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     4081 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4141 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4201 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4261 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4321 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4381 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4441 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4501 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4561 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4621 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4681 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4741 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4801 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     4861 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     4921 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4981 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5041 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5101 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5161 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5221 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5281 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5341 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     5401 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     5461 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     5521 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     5581 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     5641 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     5701 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     5761 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     5821 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     5881 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     5941 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6001 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6061 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     6121 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6181 aataggggtt ccgcgcac
//
