LOCUS       sequence-364535-        5715 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PhCMV-driven NLuc (PhCMV-NLuc-pA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5715)
  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 5715)
  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: pTS1011-Tier1-PhCMV-Nluc
FEATURES             Location/Qualifiers
     source          1..5715
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        62..441
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        442..645
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     596..616
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     690..709
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        690..708
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             761..1273
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(1328..1345)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1334..1558
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1604..2032
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1691..1710)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1901..1922
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2041..2061)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2046..2375
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2226..2361
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2288..2307
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2442..3236
                     /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(2496..2515)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3106..3125
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3410..3531
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3447..3466)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3501..3520
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3580..3596)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3580..3596)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3593..3615)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3604..3620
                     /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(3628..3634,3635..3652,3653..3658))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3673..3694
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3811..3828)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3982..4570)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4062..4081)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4741..5532,5533..5601))
                     /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     5364..5383
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5602..5706)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccgaaaagtg ccacctgacg tcgacggatc gggagatctc cacgcgtggt accctcgagt
       61 tgacattgat tattgagtag ttattaatag taatcaatta cggggtcatt agttcatagc
      121 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      181 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      241 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      301 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      361 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      421 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
      481 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
      541 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
      601 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga
      661 gaacccactg cttactggct tatcgaaatt aatacgactc actataggga gacccaagct
      721 gttcgaagcg gaattcacca tgactagtgg tggttctggt atggtcttca cactcgaaga
      781 tttcgttggg gactggcgac agacagccgg ctacaacctg gaccaagtcc ttgaacaggg
      841 aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta actccgatcc aaaggattgt
      901 cctgagcggt gaaaatgggc tgaagatcga catccatgtc atcatcccgt atgaaggtct
      961 gagcggcgac caaatgggcc agatcgaaaa aatttttaag gtggtgtacc ctgtggatga
     1021 tcatcacttt aaggtgatcc tgcactatgg cacactggta atcgacgggg ttacgccgaa
     1081 catgatcgac tatttcggac ggccgtatga aggcatcgcc gtgttcgacg gcaaaaagat
     1141 cactgtaaca gggaccctgt ggaacggcaa caaaattatc gacgagcgcc tgatcaaccc
     1201 cgacggctcc ctgctgttcc gagtaaccat caacggagtg accggctggc ggctgtgcga
     1261 acgcattctg gcggctagcg gatccaccgg tgtctagaaa gctttgaggc cggcctgcag
     1321 ggatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     1381 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     1441 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     1501 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     1561 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     1621 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     1681 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     1741 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     1801 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     1861 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     1921 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     1981 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     2041 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     2101 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     2161 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     2221 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     2281 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     2341 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     2401 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     2461 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     2521 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     2581 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     2641 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     2701 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     2761 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     2821 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     2881 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     2941 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     3001 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     3061 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     3121 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     3181 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     3241 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     3301 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     3361 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     3421 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     3481 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     3541 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     3601 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     3661 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     3721 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     3781 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     3841 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     3901 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     3961 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4021 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4081 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4141 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4201 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4261 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4321 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4381 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     4441 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     4501 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     4561 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     4621 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     4681 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     4741 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     4801 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     4861 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     4921 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     4981 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5041 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5101 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     5161 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5221 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     5281 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     5341 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     5401 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     5461 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     5521 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     5581 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     5641 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     5701 tccgcgcaca tttcc
//
