LOCUS       sequence-364567-        5170 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Tier-1 vector encoding PCREm4-driven Nluc expression 
            (PCREm4-Nluc-pA). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5170)
  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 5170)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: pVH607-Tier1-PCREm4-Nluc
FEATURES             Location/Qualifiers
     source          1..5170
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        120..151
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     CDS             203..715
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(770..787)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    776..1000
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1046..1474
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1133..1152)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1343..1364
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1483..1503)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1488..1817
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1668..1803
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1730..1749
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1884..2678
                     /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(1938..1957)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2548..2567
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2852..2973
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2889..2908)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2943..2962
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3022..3038)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3022..3038)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3035..3057)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3046..3062
                     /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(3070..3076,3077..3094,3095..3100))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3115..3136
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3253..3270)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3424..4012)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3504..3523)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4183..4974,4975..5043))
                     /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     4806..4825
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5044..5148)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cctgacgtcg acggatcggg agatctccac gcgtagcctg acgtccgaga gccgtagcct
       61 gacgtccgag ggtaccagcc tgacgtccga gagccgtagc ctgacgtccg agtactccgt
      121 agagggtata taatggaagc tcgacttcca gcttggcgag ctcttcgaag cggaattcac
      181 catgactagt ggtggttctg gtatggtctt cacactcgaa gatttcgttg gggactggcg
      241 acagacagcc ggctacaacc tggaccaagt ccttgaacag ggaggtgtgt ccagtttgtt
      301 tcagaatctc ggggtgtccg taactccgat ccaaaggatt gtcctgagcg gtgaaaatgg
      361 gctgaagatc gacatccatg tcatcatccc gtatgaaggt ctgagcggcg accaaatggg
      421 ccagatcgaa aaaattttta aggtggtgta ccctgtggat gatcatcact ttaaggtgat
      481 cctgcactat ggcacactgg taatcgacgg ggttacgccg aacatgatcg actatttcgg
      541 acggccgtat gaaggcatcg ccgtgttcga cggcaaaaag atcactgtaa cagggaccct
      601 gtggaacggc aacaaaatta tcgacgagcg cctgatcaac cccgacggct ccctgctgtt
      661 ccgagtaacc atcaacggag tgaccggctg gcggctgtgc gaacgcattc tggcggctag
      721 cggatccacc ggtgtctaga aagctttgag gccggcctgc agggatcagc ctcgactgtg
      781 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
      841 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
      901 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
      961 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     1021 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     1081 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     1141 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     1201 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     1261 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     1321 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     1381 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     1441 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     1501 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     1561 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     1621 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     1681 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     1741 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     1801 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag
     1861 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     1921 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     1981 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     2041 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     2101 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     2161 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     2221 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     2281 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     2341 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     2401 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     2461 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     2521 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     2581 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     2641 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     2701 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     2761 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     2821 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     2881 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     2941 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     3001 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     3061 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     3121 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3181 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3241 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3301 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3361 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3421 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3481 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3541 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3601 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3661 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     3721 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     3781 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3841 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     3901 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3961 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4021 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4081 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4141 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4201 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4261 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4321 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4381 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4441 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     4501 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     4561 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     4621 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     4681 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     4741 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     4801 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     4861 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     4921 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     4981 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5041 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5101 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5161 aaaagtgcca
//
