LOCUS       sequence-364604-        5907 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PhCMV-driven green fluorescent protein mTFP1 
            (PhCMV-mTFP1-pA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5907)
  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 5907)
  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: pFOX14-Tier1-PhCMV-mTFP1
FEATURES             Location/Qualifiers
     source          1..5907
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        60..439
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        440..643
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     594..614
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     688..707
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        688..706
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             759..1463
                     /codon_start=1
                     /product="monomeric teal (cyan) fluorescent protein (Ai et 
                     al., 2006)"
                     /label=mTFP1
                     /note="mammalian codon-optimized"
                     /translation="VSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDGT
                     NTINLEVKEGAPLPFSYDILTTAFAYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTFE
                     DKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKKTTGWDASTERMYVRDGVLKG
                     DVKHKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYESAV
                     ARNSTDGMDELYK"
     primer_bind     complement(1518..1535)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1524..1748
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1794..2222
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1881..1900)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2091..2112
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2231..2251)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2236..2565
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2416..2551
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2478..2497
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2632..3426
                     /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(2686..2705)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3296..3315
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3600..3721
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3637..3656)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3691..3710
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3770..3786)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3770..3786)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3783..3805)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3794..3810
                     /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(3818..3824,3825..3842,3843..3848))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3863..3884
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4001..4018)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4172..4760)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4252..4271)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4931..5722,5723..5791))
                     /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     5554..5573
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5792..5896)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gaaaagtgcc acctgacgtc gacggatcgg gagatctcca cgcgtggtac cctcgagttg
       61 acattgatta ttgagtagtt attaatagta atcaattacg gggtcattag ttcatagccc
      121 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      181 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      241 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      301 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      361 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      421 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      481 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      541 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      601 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga
      661 acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgt
      721 tcgaagcgga attcaccatg actagtggtg gttctggtgt gagcaagggc gaggagacca
      781 caatgggcgt aatcaagccc gacatgaaga tcaagctgaa gatggagggc aacgtgaatg
      841 gccacgcctt cgtgatcgag ggcgagggcg agggcaagcc ctacgacggc accaacacca
      901 tcaacctgga ggtgaaggag ggagcccccc tgcccttctc ctacgacatt ctgaccaccg
      961 cgttcgccta cggcaacagg gccttcacca agtaccccga cgacatcccc aactacttca
     1021 agcagtcctt ccccgagggc tactcttggg agcgcaccat gaccttcgag gacaagggca
     1081 tcgtgaaggt gaagtccgac atctccatgg aggaggactc cttcatctac gagatacacc
     1141 tcaagggcga gaacttcccc cccaacggcc ccgtgatgca gaagaagacc accggctggg
     1201 acgcctccac cgagaggatg tacgtgcgcg acggcgtgct gaagggcgac gtcaagcaca
     1261 agctgctgct ggagggcggc ggccaccacc gcgttgactt caagaccatc tacagggcca
     1321 agaaggcggt gaagctgccc gactatcact ttgtggacca ccgcatcgag atcctgaacc
     1381 acgacaagga ctacaacaag gtgaccgttt acgagagcgc cgtggcccgc aactccaccg
     1441 acggcatgga cgagctgtac aaggctagcg gatccaccgg tgtctagaaa gctttgaggc
     1501 cggcctgcag ggatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     1561 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     1621 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     1681 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     1741 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     1801 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     1861 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     1921 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     1981 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2041 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2101 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2161 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2221 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     2281 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     2341 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     2401 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     2461 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     2521 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     2581 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     2641 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     2701 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     2761 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     2821 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     2881 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     2941 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3001 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3061 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     3121 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     3181 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     3241 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     3301 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     3361 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     3421 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     3481 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     3541 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     3601 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     3661 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     3721 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     3781 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     3841 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     3901 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     3961 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4021 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4081 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4141 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4201 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4261 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4321 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4381 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4441 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4501 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4561 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     4621 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4681 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4741 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4801 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     4861 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     4921 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     4981 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5041 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5101 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5161 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5221 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5281 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5341 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     5401 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5461 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5521 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     5581 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     5641 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     5701 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     5761 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     5821 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5881 aaataggggt tccgcgcaca tttcccc
//
