LOCUS       sequence-364233-        6566 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PRPBSA-driven YPet in combination with a 
            marker for puromycine selection (PRPBSA-YPet-p2A-PuroR-pA).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6566)
  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 6566)
  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: pTS1024-Tier1-PRPBSA-YPet-p2A-PuroR
FEATURES             Location/Qualifiers
     source          1..6566
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1066..1085)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1504..1560
                     /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             1579..2172
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     2072..2092
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2227..2244)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2233..2457
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2503..2931
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2590..2609)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2800..2821
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2940..2960)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2945..3274
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3125..3260
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3187..3206
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3341..4135
                     /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(3395..3414)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4005..4024
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4309..4430
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4346..4365)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4400..4419
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4479..4495)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4479..4495)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4492..4514)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4503..4519
                     /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(4527..4533,4534..4551,4552..4557))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4572..4593
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4710..4727)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4881..5469)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4961..4980)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5640..6431,6432..6500))
                     /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     6263..6282
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6501..6566,1..39))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
       61 acctgacgtc gacggatcgg gagatctcca cgcgtggtac cctcgagcgt gaccctgtct
      121 cacaaaataa agtaagcccg gactgagtgc ggaaaggcgg gcctggcggg tctggactcc
      181 ccatgcgggc caccagaggc cctgcagcct tcagtcgctt gaaggggtaa tggcgcttcc
      241 actcacaaac atggcggaca gagcgtgtga acgagatgaa cagcccctca aaaatatggc
      301 cgccgaggct ggacggccgt gccccagcag caccgcctcc gcgccccacg tgatctctcg
      361 ccgggcacag cgctgaccgc ggaggtccaa ccggaagaat gtccggattg gacattcgga
      421 agagggcccg ccttccctgg ggaatctctg cgcacgcgca gaacgcttcg accaatgaaa
      481 acacaggaag ccgtccgcgc aaccgcgttg cgtcacttct gccgcccctg tttcaaggta
      541 tatagccgta gacggaactt cgcctttctc tcggccttag cgccattttt ttgggtgagt
      601 gttttttggt tcctgcgttg ggattccgtg tacaatccat agacatctga cctcggcact
      661 tagcatcatc acagcaaact aactgtagcc tttctctctt tccctgtaga aacctctgca
      721 cctagggagc tcgaattcac catgactagt ggtggttctg gtgtgagcaa aggcgaagag
      781 ctgttcaccg gcgtggtgcc catcctggtg gagctggacg gcgacgtgaa cggccacaag
      841 ttcagcgtga gcggcgaggg cgagggcgac gccacctacg gcaagctgac cctgaagctg
      901 ctgtgcacca ccggcaagct gcccgtgccc tggcccaccc tggtgaccac cctgggctac
      961 ggcgtgcagt gcttcgcccg gtaccccgac cacatgaagc agcacgactt cttcaagagc
     1021 gccatgcccg agggctacgt gcaggagcgg accatcttct tcaaggacga cggcaactac
     1081 aagacccggg ccgaggtgaa gttcgagggc gacaccctgg tgaaccggat cgagctgaag
     1141 ggcatcgact tcaaggagga cggcaacatc ctgggccaca agctggagta caactacaac
     1201 agccacaacg tgtacatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag
     1261 atccggcaca acatcgagga cggcggcgtg cagctggccg accactacca gcagaacacc
     1321 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcta ccagagcgcc
     1381 ctgttcaagg accccaacga gaagcgggac cacatggtgc tgctggagtt cctgaccgcc
     1441 gccggcatca ccgagggcat ggacgagctg tacaaggcta gtggtggttc tggtggaagc
     1501 ggagctacta acttcagcct gctgaagcag gctggtgacg tggaggagaa ccctggacct
     1561 gctagtggtg gttctggtac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     1621 gtcccccggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     1681 accgtcgacc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     1741 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     1801 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     1861 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     1921 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtgtcgcc cgaccaccag
     1981 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     2041 gtgcccgcct tcctggagac atccgcgccc cgcaacctcc ccttctacga gcggctcggc
     2101 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     2161 aagcccggtg ccgctagcgg atccaccggt gtctagaaag ctttgaggcc ggcctgcagg
     2221 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     2281 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     2341 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     2401 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     2461 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     2521 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     2581 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     2641 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     2701 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     2761 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     2821 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     2881 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg
     2941 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3001 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     3061 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     3121 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     3181 tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg
     3241 aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat
     3301 tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     3361 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     3421 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     3481 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
     3541 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     3601 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     3661 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     3721 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     3781 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     3841 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     3901 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     3961 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     4021 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     4081 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     4141 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     4201 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     4261 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     4321 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4381 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     4441 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     4501 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4561 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4621 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4681 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4741 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4801 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4861 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4921 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4981 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5041 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5101 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5161 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5221 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5281 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5341 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5401 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5461 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5521 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5581 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5641 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5701 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5761 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5821 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5881 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5941 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6001 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6061 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6121 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6181 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6241 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6301 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6361 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6421 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6481 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6541 tattgtctca tgagcggata catatt
//
