LOCUS       sequence-364973-        7520 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PTetO7-driven SEAP-p2A-iRFP670 expression 
            (OTetO7-PCMVmin-2-SEAP-p2A-iRFP670-pA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7520)
  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 7520)
  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: pVH268-Tier1-OTetO7-PCMVmin2-SEAP-p2A-iRFP670
FEATURES             Location/Qualifiers
     source          1..7520
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(20..36)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(20..36)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(33..55)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    44..60
                     /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(68..74,75..92,93..98))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    113..134
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(251..268)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(422..1010)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(502..521)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1181..1972,1973..2041))
                     /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     1804..1823
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2042..2146)
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    2228..2246
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2264..2282
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2300..2318
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2336..2354
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2372..2390
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2408..2426
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2444..2462
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2516..2540
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     2592..2611
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2592..2610
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4241..4297
                     /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             4304..5233
                     /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(5288..5305)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5294..5518
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5564..5992
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5651..5670)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5861..5882
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6001..6021)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6006..6335
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6186..6321
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6248..6267
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             6402..7196
                     /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(6456..6475)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7066..7085
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    7370..7491
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7407..7426)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7461..7480
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
       61 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
      121 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
      181 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
      241 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
      301 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      361 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      421 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      481 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      541 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      601 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      661 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      721 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      781 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      841 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      901 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      961 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     1021 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     1081 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     1141 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     1201 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1261 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1321 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1381 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1441 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1501 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1561 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1621 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1681 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1741 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1801 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1861 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1921 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1981 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     2041 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     2101 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     2161 aagtgccacc tgacgtcgac ggatcgggag atctccacgc gtccgtacac gcctaaagca
     2221 tatacgttct ctatcactga tagggagtaa actggatata cgttctctat cactgatagg
     2281 gagtaaactg tagatacgtt ctctatcact gatagggagt aaactggtca tacgttctct
     2341 atcactgata gggagtaaac tccttatacg ttctctatca ctgataggga gtaaagtctg
     2401 catacgttct ctatcactga tagggagtaa actcttcata cgttctctat cactgatagg
     2461 gagtaaactc gagtatgtcg aggtggcgtg tacggtggga ggcctatata agcagagctc
     2521 gtttagtgaa ccgtcagatc gcgagctctc tggctaacta gagaacccac tgcttactgg
     2581 cttatcgaaa ttaatacgac tcactatagg gagacccaag ctgttcgaag cggaattcac
     2641 catgactagt tggctgtggc tggtgctgct gctgctgtgc ctggccggca acgtgcaggc
     2701 cgctagtatc atccccgtgg aggaggagaa ccccgacttc tggaacagag aggccgccga
     2761 ggccctgggc gccgccaaga agctgcagcc cgcccagacc gccgccaaga acctgatcat
     2821 cttcctgggc gacggcatgg gcgtgagcac cgtgaccgcc gccagaatcc tgaagggcca
     2881 gaagaaggac aagctgggcc ccgagatccc cctggccatg gacagattcc cctacgtggc
     2941 cctgagcaag acctacaacg tggacaagca cgtgcccgac agcggcgcca ccgccaccgc
     3001 ctacctgtgc ggcgtgaagg gcaacttcca gaccatcggc ctgagcgccg ccgccagatt
     3061 caaccagtgc aacaccacca gaggcaacga ggtgatcagc gtgatgaaca gagccaagaa
     3121 ggccggcaag agcgtgggcg tggtgaccac caccagagtg cagcacgcca gccccgccgg
     3181 cacctacgcc cacaccgtga acagaaactg gtacagcgac gccgacgtgc ccgccagcgc
     3241 cagacaggag ggctgccagg acatcgccac ccagctgatc agcaacatgg acatcgacgt
     3301 gatcctgggc ggcggcagaa agtacatgtt cagaatgggc acccccgacc ccgagtaccc
     3361 cgacgactac agccagggcg gcaccagact ggacggcaag aacctggtgc aggagtggct
     3421 ggccaagaga cagggcgcca gatacgtgtg gaacagaacc gagctgatgc aggccagcct
     3481 ggaccccagc gtgacccacc tgatgggcct gttcgagccc ggcgacatga agtacgagat
     3541 ccacagagac agcaccctgg accccagcct gatggagatg accgaggccg ccctgagact
     3601 gctgagcaga aaccccagag gcttcttcct gttcgtggag ggcggcagaa tcgaccacgg
     3661 ccaccacgag agcagagcct acagagccct gaccgagacc atcatgttcg acgacgccat
     3721 cgagagagcc ggccagctga ccagcgagga ggacaccctg agcctggtga ccgccgacca
     3781 cagccacgtg ttcagcttcg gcggctaccc cctgagaggc agcagcatct tcggcctggc
     3841 ccccggcaag gccagagaca gaaaggccta caccgtgctg ctgtacggca acggccccgg
     3901 ctacgtgctg aaggacggcg ccagacccga cgtgaccgag agcgagagcg gcagccccga
     3961 gtacagacag cagagcgccg tgcccctgga cgaggagacc cacgccggcg aggacgtggc
     4021 cgtgttcgcc agaggccccc aggcccacct ggtgcacggc gtgcaggagc agaccttcat
     4081 cgcccacgtg atggccttcg ccgcctgcct ggagccctac accgcctgcg acctggcccc
     4141 ccccgccggc accaccgacg ccgcccaccc cggctacagc agagtgggcg ccgccggcag
     4201 attcgagcag accgctagtg gtggttctgg tggaagcgga gctactaact tcagcctgct
     4261 gaagcaggct ggtgacgtgg aggagaaccc tggacctgct agtgcgcgta aggtcgatct
     4321 cacctcctgc gatcgcgagc cgatccacat ccccggcagc attcagccgt gcggctgcct
     4381 gcttgcctgc gacgcgcagg cggtgcggat cacgcgcatt acggaaaatg ccggcgcgtt
     4441 ctttggacgc gaaactccgc gggtcggtga gctactcgcc gattacttcg gcgagaccga
     4501 agcccatgcg ctgcgcaacg cactggcgca gtcctccgat ccaaagcgac cggcgctgat
     4561 cttcggttgg cgcgacggcc tgaccggccg caccttcgac atctcactgc atcgccatga
     4621 cggtacatcg atcatcgagt tcgagcctgc ggcggccgaa caggccgaca atccgctgcg
     4681 gctgacgcgg cagatcatcg cgcgcaccaa agaactgaag tcgctcgaag agatggccgc
     4741 acgggtgccg cgctatctgc aggcgatgct cggctatcac cgcgtgatgt tgtaccgctt
     4801 cgcggacgac ggctccggga tggtgatcgg cgaggcgaag cgcagcgacc tcgagagctt
     4861 tctcggtcag cactttccgg cgtcgctggt cccgcagcag gcgcggctac tgtacttgaa
     4921 gaacgcgatc cgcgtggtct cggattcgcg cggcatcagc agccggatcg tgcccgagca
     4981 cgacgcctcc ggcgccgcgc tcgatctgtc gttcgcgcac ctgcgcagca tctcgccctg
     5041 ccatctcgaa tttctgcgga acatgggcgt cagcgcctcg atgtcgctgt cgatcatcat
     5101 tgacggcacg ctatggggat tgatcatctg tcatcattac gagccgcgtg ccgtgccgat
     5161 ggcgcagcgc gtcgcggccg aaatgttcgc cgacttctta tcgctgcact tcaccgccgc
     5221 ccaccaccaa cgcgctagcg gatccaccgg tgtctagaaa gctttgaggc cggcctgcag
     5281 ggatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     5341 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     5401 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     5461 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     5521 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     5581 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     5641 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     5701 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     5761 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     5821 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     5881 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     5941 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     6001 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     6061 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     6121 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     6181 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     6241 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     6301 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     6361 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     6421 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     6481 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     6541 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     6601 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     6661 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     6721 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     6781 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     6841 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     6901 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     6961 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     7021 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     7081 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     7141 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     7201 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     7261 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     7321 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     7381 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     7441 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     7501 tataccgtcg acctctagct
//
