LOCUS       sequence-366603-        7297 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PTtgO2-driven SEAP-p2A-iRFP670 expression 
            (OTtgO2-PCMVmin-2-SEAP-p2A-iRFP670-pA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7297)
  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 7297)
  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: pVH270-Tier1-OTtgO2-PCMVmin2-SEAP-p2A-iRFP670
FEATURES             Location/Qualifiers
     source          1..7297
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(49..65)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(49..65)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(62..84)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    73..89
                     /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(97..103,104..121,122..127))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    142..163
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(280..297)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(451..1039)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(531..550)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1210..2001,2002..2070))
                     /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     1833..1852
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2071..2175)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2322..2346
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     2398..2417
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2398..2416
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4047..4103
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4110..5039
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 670 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP670
                     /note="derived from Rhodopseudomonas palustris RpBphP6"
                     /translation="ARKVDLTSCDREPIHIPGSIQPCGCLLACDAQAVRITRITENAGA
                     FFGRETPRVGELLADYFGETEAHALRNALAQSSDPKRPALIFGWRDGLTGRTFDISLHR
                     HDGTSIIEFEPAAAEQADNPLRLTRQIIARTKELKSLEEMAARVPRYLQAMLGYHRVML
                     YRFADDGSGMVIGEAKRSDLESFLGQHFPASLVPQQARLLYLKNAIRVVSDSRGISSRI
                     VPEHDASGAALDLSFAHLRSISPCHLEFLRNMGVSASMSLSIIIDGTLWGLIICHHYEP
                     RAVPMAQRVAAEMFADFLSLHFTAAHHQR"
     primer_bind     complement(5094..5111)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5100..5324
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5370..5798
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5457..5476)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5667..5688
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5807..5827)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5812..6141
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5992..6127
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6054..6073
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             6208..7002
                     /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(6262..6281)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6872..6891
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    7176..7297
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7213..7232)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7267..7286
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
       61 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
      121 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
      181 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
      241 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
      301 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
      361 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
      421 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
      481 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
      541 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
      601 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
      661 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
      721 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
      781 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
      841 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
      901 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
      961 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     1021 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     1081 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     1141 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     1201 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     1261 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     1321 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     1381 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     1441 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     1501 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     1561 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     1621 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     1681 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     1741 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     1801 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     1861 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     1921 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     1981 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     2041 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     2101 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     2161 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     2221 tctccacgcg tggtaccgta tttacaaaca accatgaatg taagtatcat actctcgagt
     2281 atgtcgaggt ggcgtgtacg gtgggaggcc tatataagca gagctcgttt agtgaaccgt
     2341 cagatcgcga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa
     2401 tacgactcac tatagggaga cccaagctgt tcgaagcgga attcaccatg actagttggc
     2461 tgtggctggt gctgctgctg ctgtgcctgg ccggcaacgt gcaggccgct agtatcatcc
     2521 ccgtggagga ggagaacccc gacttctgga acagagaggc cgccgaggcc ctgggcgccg
     2581 ccaagaagct gcagcccgcc cagaccgccg ccaagaacct gatcatcttc ctgggcgacg
     2641 gcatgggcgt gagcaccgtg accgccgcca gaatcctgaa gggccagaag aaggacaagc
     2701 tgggccccga gatccccctg gccatggaca gattccccta cgtggccctg agcaagacct
     2761 acaacgtgga caagcacgtg cccgacagcg gcgccaccgc caccgcctac ctgtgcggcg
     2821 tgaagggcaa cttccagacc atcggcctga gcgccgccgc cagattcaac cagtgcaaca
     2881 ccaccagagg caacgaggtg atcagcgtga tgaacagagc caagaaggcc ggcaagagcg
     2941 tgggcgtggt gaccaccacc agagtgcagc acgccagccc cgccggcacc tacgcccaca
     3001 ccgtgaacag aaactggtac agcgacgccg acgtgcccgc cagcgccaga caggagggct
     3061 gccaggacat cgccacccag ctgatcagca acatggacat cgacgtgatc ctgggcggcg
     3121 gcagaaagta catgttcaga atgggcaccc ccgaccccga gtaccccgac gactacagcc
     3181 agggcggcac cagactggac ggcaagaacc tggtgcagga gtggctggcc aagagacagg
     3241 gcgccagata cgtgtggaac agaaccgagc tgatgcaggc cagcctggac cccagcgtga
     3301 cccacctgat gggcctgttc gagcccggcg acatgaagta cgagatccac agagacagca
     3361 ccctggaccc cagcctgatg gagatgaccg aggccgccct gagactgctg agcagaaacc
     3421 ccagaggctt cttcctgttc gtggagggcg gcagaatcga ccacggccac cacgagagca
     3481 gagcctacag agccctgacc gagaccatca tgttcgacga cgccatcgag agagccggcc
     3541 agctgaccag cgaggaggac accctgagcc tggtgaccgc cgaccacagc cacgtgttca
     3601 gcttcggcgg ctaccccctg agaggcagca gcatcttcgg cctggccccc ggcaaggcca
     3661 gagacagaaa ggcctacacc gtgctgctgt acggcaacgg ccccggctac gtgctgaagg
     3721 acggcgccag acccgacgtg accgagagcg agagcggcag ccccgagtac agacagcaga
     3781 gcgccgtgcc cctggacgag gagacccacg ccggcgagga cgtggccgtg ttcgccagag
     3841 gcccccaggc ccacctggtg cacggcgtgc aggagcagac cttcatcgcc cacgtgatgg
     3901 ccttcgccgc ctgcctggag ccctacaccg cctgcgacct ggcccccccc gccggcacca
     3961 ccgacgccgc ccaccccggc tacagcagag tgggcgccgc cggcagattc gagcagaccg
     4021 ctagtggtgg ttctggtgga agcggagcta ctaacttcag cctgctgaag caggctggtg
     4081 acgtggagga gaaccctgga cctgctagtg cgcgtaaggt cgatctcacc tcctgcgatc
     4141 gcgagccgat ccacatcccc ggcagcattc agccgtgcgg ctgcctgctt gcctgcgacg
     4201 cgcaggcggt gcggatcacg cgcattacgg aaaatgccgg cgcgttcttt ggacgcgaaa
     4261 ctccgcgggt cggtgagcta ctcgccgatt acttcggcga gaccgaagcc catgcgctgc
     4321 gcaacgcact ggcgcagtcc tccgatccaa agcgaccggc gctgatcttc ggttggcgcg
     4381 acggcctgac cggccgcacc ttcgacatct cactgcatcg ccatgacggt acatcgatca
     4441 tcgagttcga gcctgcggcg gccgaacagg ccgacaatcc gctgcggctg acgcggcaga
     4501 tcatcgcgcg caccaaagaa ctgaagtcgc tcgaagagat ggccgcacgg gtgccgcgct
     4561 atctgcaggc gatgctcggc tatcaccgcg tgatgttgta ccgcttcgcg gacgacggct
     4621 ccgggatggt gatcggcgag gcgaagcgca gcgacctcga gagctttctc ggtcagcact
     4681 ttccggcgtc gctggtcccg cagcaggcgc ggctactgta cttgaagaac gcgatccgcg
     4741 tggtctcgga ttcgcgcggc atcagcagcc ggatcgtgcc cgagcacgac gcctccggcg
     4801 ccgcgctcga tctgtcgttc gcgcacctgc gcagcatctc gccctgccat ctcgaatttc
     4861 tgcggaacat gggcgtcagc gcctcgatgt cgctgtcgat catcattgac ggcacgctat
     4921 ggggattgat catctgtcat cattacgagc cgcgtgccgt gccgatggcg cagcgcgtcg
     4981 cggccgaaat gttcgccgac ttcttatcgc tgcacttcac cgccgcccac caccaacgcg
     5041 ctagcggatc caccggtgtc tagaaagctt tgaggccggc ctgcagggat cagcctcgac
     5101 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     5161 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     5221 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     5281 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     5341 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     5401 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     5461 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     5521 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     5581 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     5641 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     5701 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     5761 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     5821 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     5881 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     5941 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     6001 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     6061 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     6121 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     6181 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     6241 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     6301 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     6361 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     6421 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     6481 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     6541 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     6601 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     6661 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     6721 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     6781 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     6841 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     6901 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     6961 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     7021 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     7081 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     7141 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     7201 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     7261 ctagttgtgg tttgtccaaa ctcatcaatg tatctta
//
