LOCUS       sequence-364539-        6066 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PmPGK1-driven tTA expression 
            (PmPGK1-tTA-pA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6066)
  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 6066)
  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: pVH234-Tier1-PmPGK1-tTA
FEATURES             Location/Qualifiers
     source          1..6066
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(166..754)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(246..265)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(925..1716,1717..1785))
                     /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     1548..1567
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1786..1890)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1964..2463
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1999..2022)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2396..2415
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             join(2516..3133,3134..3517)
                     /codon_start=1
                     /product="tetracycline-controlled transactivator, 
                     comprising a fusion of the tetracycline repressor TetR with
                     the C-terminal activation domain of herpes simplex virus 
                     VP16"
                     /label=tTA
                     /note="In the Tet-Off(R) system, tTA binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="SRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVK
                     NKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRP
                     TEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTD
                     SMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIE
                     GLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHAD
                     ALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     primer_bind     complement(2586..2605)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3578..3595)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3584..3808
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3854..4282
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3941..3960)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4151..4172
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4291..4311)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4296..4625
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4476..4611
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4538..4557
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4692..5486
                     /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(4746..4765)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5356..5375
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5660..5781
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5697..5716)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5751..5770
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5830..5846)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5830..5846)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5843..5865)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5854..5870
                     /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(5878..5884,5885..5902,5903..5908))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5923..5944
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(join(6061..6066,1..12))
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
       61 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
      121 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
      181 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
      241 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
      301 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
      361 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
      421 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
      481 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
      541 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
      601 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
      661 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
      721 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
      781 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
      841 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
      901 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
      961 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     1021 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     1081 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     1141 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     1201 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     1261 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     1321 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     1381 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     1441 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     1501 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     1561 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     1621 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     1681 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     1741 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     1801 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     1861 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     1921 cgacggatcg ggagatctcc acgcgtggta ccctcgagct accgggtagg ggaggcgctt
     1981 ttcccaaggc agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca
     2041 agtggcctct ggcctcgcac acattccaca tccaccggta ggcgccaacc ggctccgttc
     2101 tttggtggcc ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc
     2161 ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag cacgtctcag tagtctcgtg
     2221 cagatggaca gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag
     2281 cagctttgct ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg
     2341 ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc
     2401 tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt
     2461 tcgacctcca gcccgagctc ggtataggtt cgaagcggaa ttcaccatga ctagttccag
     2521 attagataaa agtaaagtga ttaacagcgc attagagctg cttaatgagg tcggaatcga
     2581 aggtttaaca acccgtaaac tcgcccagaa gctaggtgta gagcagccta cattgtattg
     2641 gcatgtaaaa aataagcggg ctttgctcga cgccttagcc attgagatgt tagataggca
     2701 ccatactcac ttttgccctt tagaagggga aagctggcaa gattttttac gtaataacgc
     2761 taaaagtttt agatgtgctt tactaagtca tcgcgatgga gcaaaagtac atttaggtac
     2821 acggcctaca gaaaaacagt atgaaactct cgaaaatcaa ttagcctttt tatgccaaca
     2881 aggtttttca ctagagaatg cattatatgc actcagcgct gtggggcatt ttactttagg
     2941 ttgcgtattg gaagatcaag agcatcaagt cgctaaagaa gaaagggaaa cacctactac
     3001 tgatagtatg ccgccattat tacgacaagc tatcgaatta tttgatcacc aaggtgcaga
     3061 gccagccttc ttattcggcc ttgaattgat catatgcgga ttagaaaaac aacttaaatg
     3121 tgaaagtggg tccgcgtaca gccgcgcgcg tacgaaaaac aattacgggt ctaccatcga
     3181 gggcctgctc gatctcccgg acgacgacgc ccccgaagag gcggggctgg cggctccgcg
     3241 cctgtccttt ctccccgcgg gacacacgcg cagactgtcg acggcccccc cgaccgatgt
     3301 cagcctgggg gacgagctcc acttagacgg cgaggacgtg gcgatggcgc atgccgacgc
     3361 gctagacgat ttcgatctgg acatgttggg ggacggggat tccccgggtc cgggatttac
     3421 cccccacgac tccgccccct acggcgctct ggatatggcc gacttcgagt ttgagcagat
     3481 gtttaccgat gcccttggaa ttgacgagta cggtggggct agtgctagcg gatccaccgg
     3541 tgtctagaaa gctttgaggc cggcctgcag ggatcagcct cgactgtgcc ttctagttgc
     3601 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     3661 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     3721 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     3781 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     3841 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3901 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3961 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4021 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     4081 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4141 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     4201 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     4261 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     4321 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     4381 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     4441 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     4501 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     4561 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     4621 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     4681 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     4741 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     4801 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     4861 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     4921 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     4981 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     5041 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     5101 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     5161 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     5221 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     5281 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     5341 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     5401 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     5461 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     5521 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     5581 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     5641 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     5701 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     5761 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     5821 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     5881 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     5941 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     6001 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     6061 ctcgct
//
