LOCUS       sequence-364599-        5895 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Tier-1 vector encoding PhCMV-driven green fluorescent protein turbo 
            GFP (PhCMV-tGFP-pA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5895)
  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 5895)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pFOX13-Tier1-PhCMV-tGFP
FEATURES             Location/Qualifiers
     source          1..5895
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        59..438
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        439..642
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     593..613
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     687..706
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        687..705
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             758..1450
                     /codon_start=1
                     /product="improved green fluorescent protein from 
                     Pontellina plumata (Evdokimov et al., 2006)"
                     /label=TurboGFP
                     /note="mammalian codon-optimized"
                     /translation="ESDESGLPAMEIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMK
                     STKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLH
                     VSFSYRYEAGRVIGDFKVMGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNDLDGSFTRT
                     FSLRDGGYYSSVVDSHMHFKSAIHPSILQNGGPMFAFRRVEEDHSNTELGIVEYQHAFK
                     TPDADAGEE"
     primer_bind     complement(1505..1522)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1511..1735
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1781..2209
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1868..1887)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2078..2099
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2218..2238)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2223..2552
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2403..2538
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2465..2484
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2619..3413
                     /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(2673..2692)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3283..3302
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3587..3708
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3624..3643)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3678..3697
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3757..3773)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3757..3773)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3770..3792)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3781..3797
                     /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(3805..3811,3812..3829,3830..3835))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3850..3871
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3988..4005)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4159..4747)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4239..4258)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4918..5709,5710..5778))
                     /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     5541..5560
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5779..5883)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaaagtgcca cctgacgtcg acggatcggg agatctccac gcgtggtacc ctcgagttga
       61 cattgattat tgagtagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
      121 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      181 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      241 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      301 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      361 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      421 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      481 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      541 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      601 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa
      661 cccactgctt actggcttat cgaaattaat acgactcact atagggagac ccaagctgtt
      721 cgaagcggaa ttcaccatga ctagtggtgg ttctggtgag agcgacgaga gcggcctgcc
      781 cgccatggag atcgagtgcc gcatcaccgg caccctgaac ggcgtggagt tcgagctggt
      841 gggcggcgga gagggcaccc ccgagcaggg ccgcatgacc aacaagatga agagcaccaa
      901 aggcgccctg accttcagcc cctacctgct gagccacgtg atgggctacg gcttctacca
      961 cttcggcacc taccccagcg gctacgagaa ccccttcctg cacgccatca acaacggcgg
     1021 ctacaccaac acccgcatcg agaagtacga ggacggcggc gtgctgcacg tgagcttcag
     1081 ctaccgctac gaggccggcc gcgtgatcgg cgacttcaag gtgatgggca ccggcttccc
     1141 cgaggacagc gtgatcttca ccgacaagat catccgcagc aacgccaccg tggagcacct
     1201 gcaccccatg ggcgataacg atctggatgg cagcttcacc cgcaccttca gcctgcgcga
     1261 cggcggctac tacagctccg tggtggacag ccacatgcac ttcaagagcg ccatccaccc
     1321 cagcatcctg cagaacgggg gccccatgtt cgccttccgc cgcgtggagg aggatcacag
     1381 caacaccgag ctgggcatcg tggagtacca gcacgccttc aagaccccgg atgcagatgc
     1441 cggtgaagaa gctagcggat ccaccggtgt ctagaaagct ttgaggccgg cctgcaggga
     1501 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     1561 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     1621 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     1681 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     1741 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     1801 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     1861 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     1921 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     1981 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2041 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     2101 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     2161 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
     2221 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     2281 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     2341 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     2401 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     2461 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     2521 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     2581 tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     2641 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     2701 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     2761 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     2821 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     2881 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     2941 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     3001 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     3061 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     3121 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     3181 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     3241 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     3301 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     3361 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     3421 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     3481 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     3541 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     3601 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     3661 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     3721 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     3781 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     3841 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     3901 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3961 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4021 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4081 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4141 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4201 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4261 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4321 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4381 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4441 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4501 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4561 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     4621 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4681 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     4741 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4801 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4861 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4921 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4981 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5041 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     5101 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5161 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5221 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5281 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5341 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5401 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     5461 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     5521 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5581 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     5641 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     5701 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     5761 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     5821 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5881 gcgcacattt ccccg
//
