LOCUS       sequence-366205-        5802 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PmPGK1-driven rtTA expression 
            (PmPGK1-rtTA-pA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5802)
  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 5802)
  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: pVH235-Tier1-PmPGK1-rtTA
FEATURES             Location/Qualifiers
     source          1..5802
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1..18)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(172..760)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(252..271)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(931..1722,1723..1791))
                     /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     1554..1573
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1792..1896)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1970..2469
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2005..2028)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2402..2421
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2525..3265
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="SRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVK
                     NKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRP
                     TEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTD
                     SMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPAD
                     ALDDFDLDMLPADALDDFDLDMLPG"
     primer_bind     complement(3320..3337)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3326..3550
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3596..4024
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3683..3702)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3893..3914
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4033..4053)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4038..4367
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4218..4353
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4280..4299
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4434..5228
                     /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(4488..4507)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5098..5117
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5402..5523
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5439..5458)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5493..5512
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5572..5588)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5572..5588)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5585..5607)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5596..5612
                     /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(5620..5626,5627..5644,5645..5650))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5665..5686
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
       61 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
      121 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
      181 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
      241 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
      301 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
      361 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
      421 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
      481 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
      541 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
      601 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
      661 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
      721 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
      781 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
      841 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
      901 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
      961 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     1021 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     1081 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     1141 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     1201 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     1261 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     1321 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     1381 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     1441 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     1501 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     1561 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     1621 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     1681 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     1741 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     1801 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     1861 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     1921 tgacgtcgac ggatcgggag atctccacgc gtggtaccct cgagctaccg ggtaggggag
     1981 gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg cacttggcgc
     2041 tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct
     2101 ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc
     2161 cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcagtagt
     2221 ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc
     2281 caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg tgggtccggg
     2341 ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc cggaggcccg
     2401 gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg
     2461 ggcctttcga cctccagccc gagctcggta taggttcgaa gcggaattca ccatgactag
     2521 tggatcaaga ctggacaaga gcaaagtcat aaactctgct ctggaattac tcaatggagt
     2581 cggtatcgaa ggcctgacga caaggaaact cgctcaaaag ctgggagttg agcagcctac
     2641 cctgtactgg cacgtgaaga acaagcgggc cctgctcgat gccctgccaa tcgagatgct
     2701 ggacaggcat catacccact cctgccccct ggaaggcgag tcatggcaag actttctgcg
     2761 gaacaacgcc aagtcatacc gctgtgctct cctctcacat cgcgacgggg ctaaagtgca
     2821 tctcggcacc cgcccaacag agaaacagta cgaaaccctg gaaaatcagc tcgcgttcct
     2881 gtgtcagcaa ggcttctccc tggagaacgc actgtacgct ctgtccgccg tgggccactt
     2941 tacactgggc tgcgtattgg aggaacagga gcatcaagta gcaaaagagg aaagagagac
     3001 acctaccacc gattctatgc ccccacttct gaaacaagca attgagctgt tcgaccggca
     3061 gggagccgaa cctgccttcc ttttcggcct ggaactaatc atatgtggcc tggagaaaca
     3121 gctaaagtgc gaaagcggcg ggccgaccga cgcccttgac gattttgact tagacatgct
     3181 cccagccgat gcccttgacg actttgacct tgatatgctg cctgctgacg ctcttgacga
     3241 ttttgacctt gacatgctcc ccggggctag cggatccacc ggtgtctaga aagctttgag
     3301 gccggcctgc agggatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     3361 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     3421 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     3481 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     3541 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     3601 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3661 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3721 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3781 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3841 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3901 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3961 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     4021 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     4081 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     4141 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     4201 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     4261 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     4321 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     4381 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     4441 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     4501 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     4561 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     4621 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     4681 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     4741 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     4801 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     4861 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     4921 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     4981 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     5041 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     5101 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     5161 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     5221 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     5281 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     5341 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     5401 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     5461 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     5521 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     5581 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     5641 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5701 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5761 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tc
//
