LOCUS       sequence-364592-        5916 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PhCMV-driven gren fluorescent protein clover 
            (PhCMV-clover-pA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5916)
  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 5916)
  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: pFOX11-Tier1-PhCMV-Clover
FEATURES             Location/Qualifiers
     source          1..5916
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        40..419
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        420..623
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     574..594
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     668..687
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        668..686
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(739..741,742..1452)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(781..802)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1389..1410
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1507..1524)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1513..1737
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1783..2211
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1870..1889)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2080..2101
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2220..2240)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2225..2554
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2405..2540
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2467..2486
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2621..3415
                     /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(2675..2694)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3285..3304
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3589..3710
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3626..3645)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3680..3699
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3759..3775)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3759..3775)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3772..3794)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3783..3799
                     /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(3807..3813,3814..3831,3832..3837))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3852..3873
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3990..4007)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4161..4749)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4241..4260)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4920..5711,5712..5780))
                     /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     5543..5562
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5781..5885)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctcca cgcgtggtac cctcgagttg acattgatta ttgagtagtt
       61 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
      121 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
      181 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
      241 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
      301 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
      361 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
      421 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
      481 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
      541 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
      601 gggaggtcta tataagcaga gctctctggc taactagaga acccactgct tactggctta
      661 tcgaaattaa tacgactcac tatagggaga cccaagctgt tcgaagcgga attcaccatg
      721 actagtggtg gttctggtgt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
      781 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtccgcgg cgagggcgag
      841 ggcgatgcca ccaacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
      901 gtgccctggc ccaccctcgt gaccaccttc ggctacggcg tggcctgctt cagccgctac
      961 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     1021 gagcgcacca tctctttcaa ggacgacggt acctacaaga cccgcgccga ggtgaagttc
     1081 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     1141 aacatcctgg ggcacaagct ggagtacaac ttcaacagcc acaacgtcta tatcacggcc
     1201 gacaagcaga agaacggcat caaggctaac ttcaagatcc gccacaacgt tgaggacggc
     1261 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     1321 ctgcccgaca accactacct gagccatcag tccgccctga gcaaagaccc caacgagaag
     1381 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggattacaca tggcatggac
     1441 gagctgtaca aggctagcgg atccaccggt gtctagaaag ctttgaggcc ggcctgcagg
     1501 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     1561 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     1621 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     1681 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     1741 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     1801 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     1861 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     1921 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     1981 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     2041 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     2101 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     2161 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg
     2221 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     2281 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     2341 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     2401 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     2461 tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg
     2521 aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat
     2581 tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     2641 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     2701 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     2761 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
     2821 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     2881 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     2941 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     3001 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     3061 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     3121 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     3181 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     3241 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     3301 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     3361 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     3421 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     3481 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     3541 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     3601 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     3661 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     3721 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     3781 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     3841 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     3901 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     3961 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4021 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4081 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4141 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4201 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4261 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4321 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4381 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4441 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4501 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4561 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4621 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4681 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4741 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4801 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     4861 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     4921 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4981 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5041 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5101 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5161 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5221 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5281 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5341 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5401 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5461 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5521 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5581 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5641 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5701 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5761 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     5821 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5881 ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
