LOCUS       sequence-364207-        5061 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PPGK-controlled 1x (AEAAAK)-linker 
            (PPGK-AEAAAK-pA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5061)
  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 5061)
  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: pTS2415-Tier1-PPGK-1xEAAAK
FEATURES             Location/Qualifiers
     source          1..5061
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(32..49)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(203..791)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(283..302)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(962..1753,1754..1822))
                     /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     1585..1604
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1823..1927)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2001..2488
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2036..2059)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2421..2440
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     complement(2610..2627)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2616..2840
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2886..3314
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2973..2992)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3183..3204
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3323..3343)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3328..3657
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3508..3643
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3570..3589
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3724..4518
                     /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(3778..3797)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4388..4407
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4692..4813
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4729..4748)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4783..4802
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4862..4878)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4862..4878)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4875..4897)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4886..4902
                     /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(4910..4916,4917..4934,4935..4940))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4955..4976
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
       61 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
      121 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
      181 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
      241 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
      301 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
      361 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
      421 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
      481 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
      541 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
      601 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
      661 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
      721 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
      781 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
      841 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
      901 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
      961 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     1021 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     1081 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     1141 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     1201 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     1261 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     1321 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     1381 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     1441 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     1501 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     1561 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     1621 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     1681 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     1741 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     1801 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     1861 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     1921 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctccacg
     1981 cgtggtaccc tcgagctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg
     2041 cgctttagca gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca
     2101 ttccacatcc accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc
     2161 ttctactcct cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac
     2221 gtgacaaatg gaagtagcac gtctcagtag tctcgtgcag atggacagca ccgctgagca
     2281 atggaagcgg gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg
     2341 ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc aggggcgggg cgggcgcccg
     2401 aaggtcctcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt
     2461 ctcctcttcc tcatctccgg gcctttcgac ctccagcccg agctcggtat agggagaccg
     2521 aattcaccat gactagtgcc gaagccgccg cgaaggctag cggatccacc ggtgtctaga
     2581 aagctttgag gccggcctgc agggatcagc ctcgactgtg ccttctagtt gccagccatc
     2641 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     2701 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     2761 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     2821 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     2881 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2941 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3001 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3061 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     3121 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     3181 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     3241 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     3301 atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     3361 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     3421 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     3481 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     3541 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     3601 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     3661 ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt
     3721 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     3781 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     3841 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     3901 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     3961 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     4021 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     4081 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     4141 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     4201 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     4261 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     4321 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     4381 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     4441 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     4501 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     4561 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     4621 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     4681 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4741 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4801 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     4861 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     4921 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     4981 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5041 tcggccaacg cgcggggaga g
//
