LOCUS       sequence-364232-        6612 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PhCMV-driven YPet in combination with a 
            marker for puromycin selection (PhCMV-YPet-p2A-PuroR-pA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6612)
  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 6612)
  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: pTS1047-Tier1-PhCMV-YPet-p2A-PuroR
FEATURES             Location/Qualifiers
     source          1..6612
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    8..129
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(45..64)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     99..118
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(178..194)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(178..194)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(191..213)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    202..218
                     /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(226..232,233..250,251..256))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    271..292
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(409..426)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(580..1168)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(660..679)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1339..2130,2131..2199))
                     /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     1962..1981
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2200..2304)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        2375..2754
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2755..2958
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2909..2929
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3003..3022
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3003..3021
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3377..3396)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             3815..3871
                     /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             3890..4483
                     /codon_start=1
                     /gene="pac from Streptomyces"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVECPKDRATWCMTRKPGA"
     primer_bind     4383..4403
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4538..4555)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4544..4768
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4814..5242
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4901..4920)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5111..5132
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5251..5271)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5256..5585
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5436..5571
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5498..5517
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5652..6446
                     /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(5706..5725)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6316..6335
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
       61 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
      121 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
      181 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
      241 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
      301 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
      361 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
      421 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
      481 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
      541 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
      601 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
      661 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
      721 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
      781 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
      841 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
      901 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
      961 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     1021 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     1081 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     1141 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     1201 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     1261 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     1321 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     1381 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     1441 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     1501 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     1561 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     1621 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     1681 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     1741 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     1801 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     1861 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     1921 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     1981 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     2041 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     2101 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     2161 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     2221 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     2281 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg
     2341 atcgggagat ctccacgcgt ggtaccctcg agttgacatt gattattgag tagttattaa
     2401 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     2461 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     2521 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     2581 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     2641 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     2701 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
     2761 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
     2821 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
     2881 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
     2941 gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa
     3001 attaatacga ctcactatag ggagacccaa gctgttcgaa gcggaattca ccatgactag
     3061 tggtggttct ggtgtgagca aaggcgaaga gctgttcacc ggcgtggtgc ccatcctggt
     3121 ggagctggac ggcgacgtga acggccacaa gttcagcgtg agcggcgagg gcgagggcga
     3181 cgccacctac ggcaagctga ccctgaagct gctgtgcacc accggcaagc tgcccgtgcc
     3241 ctggcccacc ctggtgacca ccctgggcta cggcgtgcag tgcttcgccc ggtaccccga
     3301 ccacatgaag cagcacgact tcttcaagag cgccatgccc gagggctacg tgcaggagcg
     3361 gaccatcttc ttcaaggacg acggcaacta caagacccgg gccgaggtga agttcgaggg
     3421 cgacaccctg gtgaaccgga tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     3481 cctgggccac aagctggagt acaactacaa cagccacaac gtgtacatca ccgccgacaa
     3541 gcagaagaac ggcatcaagg ccaacttcaa gatccggcac aacatcgagg acggcggcgt
     3601 gcagctggcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     3661 cgacaaccac tacctgagct accagagcgc cctgttcaag gaccccaacg agaagcggga
     3721 ccacatggtg ctgctggagt tcctgaccgc cgccggcatc accgagggca tggacgagct
     3781 gtacaaggct agtggtggtt ctggtggaag cggagctact aacttcagcc tgctgaagca
     3841 ggctggtgac gtggaggaga accctggacc tgctagtggt ggttctggta ccgagtacaa
     3901 gcccacggtg cgcctcgcca cccgcgacga cgtcccccga gccgtacgca ccctcgccgc
     3961 cgcgttcgcc gactaccccg ccacgcgcca caccgtcgac ccggaccgcc acatcgagcg
     4021 ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg
     4081 ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg
     4141 ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc
     4201 gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct
     4261 ggccaccgtc ggcgtgtcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct
     4321 ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga catccgcgcc
     4381 ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgagtgccc
     4441 gaaggaccgc gcgacctggt gcatgacccg caagcccggt gccgctagcg gatccaccgg
     4501 tgtctagaaa gctttgaggc cggcctgcag ggatcagcct cgactgtgcc ttctagttgc
     4561 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     4621 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     4681 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     4741 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     4801 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4861 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     4921 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4981 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     5041 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     5101 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     5161 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     5221 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     5281 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     5341 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     5401 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     5461 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     5521 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     5581 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     5641 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     5701 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     5761 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     5821 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     5881 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     5941 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     6001 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     6061 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     6121 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     6181 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     6241 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     6301 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     6361 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     6421 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     6481 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     6541 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     6601 ggagttcttc gc
//
