LOCUS       sequence-364247-        5916 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PhCMV-driven green fluorescent protein eGFP 
            (PhCMV-eGFP-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: pFOX12-Tier1-PhCMV-eGFP
FEATURES             Location/Qualifiers
     source          1..5916
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        48..427
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        428..631
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     582..602
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     676..695
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        676..694
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(747..749,750..1460)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(747..749,750..1460)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(789..810)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1050..1069)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1397..1418
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1515..1532)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1521..1745
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1791..2219
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1878..1897)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2088..2109
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2228..2248)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2233..2562
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2413..2548
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2475..2494
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2629..3423
                     /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(2683..2702)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3293..3312
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3597..3718
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3634..3653)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3688..3707
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3767..3783)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3767..3783)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3780..3802)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3791..3807
                     /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(3815..3821,3822..3839,3840..3845))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3860..3881
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3998..4015)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4169..4757)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4249..4268)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4928..5719,5720..5788))
                     /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     5551..5570
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5789..5893)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgtcga cggatcggga gatctccacg cgtggtaccc tcgagttgac attgattatt
       61 gagtagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
      121 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
      181 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
      241 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      301 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
      361 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      421 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
      481 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
      541 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
      601 tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac ccactgctta
      661 ctggcttatc gaaattaata cgactcacta tagggagacc caagctgttc gaagcggaat
      721 tcaccatgac tagtggtggt tctggtgtga gcaagggcga ggagctgttc accggggtgg
      781 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
      841 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
      901 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
      961 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     1021 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     1081 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     1141 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     1201 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     1261 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     1321 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     1381 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     1441 gcatggacga gctgtacaag gctagcggat ccaccggtgt ctagaaagct ttgaggccgg
     1501 cctgcaggga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     1561 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     1621 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     1681 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     1741 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     1801 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     1861 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     1921 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     1981 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     2041 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     2101 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     2161 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
     2221 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     2281 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     2341 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     2401 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     2461 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
     2521 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
     2581 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     2641 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     2701 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     2761 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     2821 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     2881 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     2941 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     3001 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     3061 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     3121 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     3181 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     3241 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     3301 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     3361 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     3421 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     3481 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     3541 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     3601 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     3661 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     3721 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     3781 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3841 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3901 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     3961 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4021 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4081 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4141 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4201 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4261 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4321 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4381 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4441 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4501 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4561 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4621 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4681 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4741 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4801 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4861 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4921 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4981 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5041 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5101 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5161 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5221 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5281 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5341 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5401 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5461 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5521 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5581 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5641 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5701 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5761 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5821 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5881 taggggttcc gcgcacattt ccccgaaaag tgccac
//
