LOCUS       sequence-364228-        5880 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PhCMV-driven p2A sequence fused to a marker 
            for puromycine selection (PhCMV-p2A-PuroR-pA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5880)
  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 5880)
  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: pTS1023-Tier1-PhCMV-p2A-PuroR
FEATURES             Location/Qualifiers
     source          1..5880
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(22..41)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     76..95
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(155..171)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(155..171)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(168..190)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    179..195
                     /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(203..209,210..227,228..233))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    248..269
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(386..403)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(557..1145)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(637..656)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1316..2107,2108..2176))
                     /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     1939..1958
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2177..2281)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        2352..2731
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2732..2935
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2886..2906
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2980..2999
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2980..2998
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3060..3116
                     /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             3135..3728
                     /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     3628..3648
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3783..3800)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3789..4013
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4059..4487
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4146..4165)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4356..4377
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4496..4516)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4501..4830
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4681..4816
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4743..4762
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4897..5691
                     /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(4951..4970)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5561..5580
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    join(5865..5880,1..106)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
       61 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
      121 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
      181 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
      241 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
      301 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
      361 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
      421 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
      481 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
      541 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
      601 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
      661 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
      721 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
      781 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
      841 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
      901 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
      961 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     1021 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     1081 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     1141 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     1201 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     1261 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     1321 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     1381 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     1441 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     1501 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     1561 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     1621 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     1681 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     1741 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     1801 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     1861 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     1921 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     1981 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     2041 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     2101 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     2161 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     2221 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     2281 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc cacgcgtggt
     2341 accctcgagt tgacattgat tattgagtag ttattaatag taatcaatta cggggtcatt
     2401 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     2461 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     2521 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     2581 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     2641 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     2701 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
     2761 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     2821 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     2881 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg
     2941 gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc actataggga
     3001 gacccaagct gttcgaagcg gaattcacca tgactagtgg tggttctggt ggaagcggag
     3061 ctactaactt cagcctgctg aagcaggctg gtgacgtgga ggagaaccct ggacctgcta
     3121 gtggtggttc tggtaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc
     3181 cccgggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg
     3241 tcgacccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg
     3301 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga
     3361 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg
     3421 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc
     3481 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt gtcgcccgac caccagggca
     3541 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc
     3601 ccgccttcct ggagacatcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca
     3661 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc
     3721 ccggtgccgc tagcggatcc accggtgtct agaaagcttt gaggccggcc tgcagggatc
     3781 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     3841 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     3901 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     3961 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     4021 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     4081 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     4141 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4201 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4261 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     4321 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     4381 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     4441 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     4501 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4561 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     4621 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     4681 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     4741 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     4801 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     4861 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     4921 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     4981 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     5041 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     5101 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     5161 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     5221 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     5281 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     5341 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     5401 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     5461 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     5521 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     5581 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     5641 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     5701 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     5761 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     5821 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
//
