LOCUS       sequence-39433-e        7940 bp    DNA     circular SYN 14-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7940)
  AUTHORS   Alexeyev MF, Fayzulin R, Shokolenko IN, Pastukh V
  TITLE     A retro-lentiviral system for doxycycline-inducible gene expression 
            and gene knockdown in cells with limited proliferative capacity.
  JOURNAL   Mol Biol Rep. 2010 Apr . 37(4):1987-91.
  PUBMED    19655272
REFERENCE   2  (bases 1 to 7940)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Biol 
            Rep. 2010 Apr . 37(4):1987-91."
COMMENT     Sequence Label: pMA2641
FEATURES             Location/Qualifiers
     source          1..7940
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..517
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     CDS             join(1074..1691,1692..1697,1698..1733,1734..1736,1737..1772,
                     1773..1775,1776..1811,1812..1820)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    join(1844..2417,2418..2420,2421..2429)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2011..2028)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2238..2257
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2430..2432,2433..2435,2436..3143)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     CDS             join(2430..2432,2433..2435,2436..3143)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     primer_bind     complement(2475..2496)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2736..2755)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3083..3104
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      3147..3156
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3153..3551
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    3627..4215
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3680..3700)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4098..4109)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             4223..4240
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5554..5573)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5673..5695
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5733..5751)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5819..5923
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5924..5992,5993..6784)
                     /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     complement(6142..6161)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6955..7543
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7444..7463
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7697..7714
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc
       61 atggaaaata cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag
      121 cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccgct cagggccaag
      181 aacagatggt ccccagatgc ggtcccgccc tcagcagttt ctagagaacc atcagatgtt
      241 tccagggtgc cccaaggacc tgaaaatgac cctgtgcctt atttgaacta accaatcagt
      301 tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac
      361 ccctcactcg gcgcgccagt cctccgattg actgcgtcgc ccgggtaccc gtattcccaa
      421 taaagcctct tgctgtttgc atccgaatcg tggactcgct gatccttggg agggtctcct
      481 cagattgatt gactgcccac ctcgggggtc tttcatttgg aggttccacc gagatttgga
      541 gacccctgcc cagggaccac cgaccccccc gccgggaggt aagctggcca gcggtcgttt
      601 cgtgtctgtc tctgtctttg ggcgtgtttg tgccggcatc tagtgtttgc gcctgcgtct
      661 gtactagttg gctaactaga tctgtatctg gcggtcccgc ggaagaactg acgagttcgt
      721 attcccggcc gcagcccctg ggagacgtcc cagcggcctc gggggcccgt tttgtggccc
      781 attctgtatc agttaaccta cccgagtcgg actttttgga gctccgccac tgtccgaggg
      841 gtacgtggct ttgttggggg acgagagaca gagacacttc gcgcccccgt ctgaattttt
      901 gctttcggtt ttacgccgaa accgcgccgc gcgtcttgtc tgctgcagca tcgttctgtg
      961 ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat tagctcgaca aagttaagta
     1021 atagtccctc tctccaagct cacttacagg cggccgggct gcaggaattc accatgtcta
     1081 gactggacaa gagcaaagtc ataaacggcg ctctggaatt actcaatgga gtcggtatcg
     1141 aaggcctgac gacaaggaaa ctcgctcaaa agctgggagt tgagcagcct accctgtact
     1201 ggcacgtgaa gaacaagcgg gccctgctcg atgccctgcc aatcgagatg ctggacaggc
     1261 atcataccca cttctgcccc ctggaaggcg agtcatggca agactttctg cggaacaacg
     1321 ccaagtcatt ccgctgtgct ctcctctcac atcgcgacgg ggctaaagtg catctcggca
     1381 cccgcccaac agagaaacag tacgaaaccc tggaaaatca gctcgcgttc ctgtgtcagc
     1441 aaggcttctc cctggagaac gcactgtacg ctctgtccgc cgtgggccac tttacactgg
     1501 gctgcgtatt ggaggaacag gagcatcaag tagcaaaaga ggaaagagag acacctacca
     1561 ccgattctat gcccccactt ctgagacaag caattgagct gttcgaccgg cagggagccg
     1621 aacctgcctt ccttttcggc ctggaactaa tcatatgtgg cctggagaaa cagctaaagt
     1681 gcgaaagcgg cgggccggcc gacgcccttg acgattttga cttagacatg ctcccagccg
     1741 atgcccttga cgactttgac cttgatatgc tgcctgctga cgctcttgac gattttgacc
     1801 ttgacatgct ccccgggtaa ctaagtaagg atcccccggc cgccccctct ccctcccccc
     1861 cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt
     1921 tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct
     1981 tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga
     2041 atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga
     2101 ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac
     2161 gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag
     2221 ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc
     2281 agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg
     2341 tttagtcgag gttaaaaaac gtctaggccc cccgaaccac ggggacgtgg tttccctttg
     2401 aaaaacacga tgattatatg gccacaacca tggtgagcaa gggcgaggag ctgttcaccg
     2461 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     2521 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     2581 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     2641 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     2701 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     2761 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     2821 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     2881 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     2941 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     3001 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     3061 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     3121 ctctcggcat ggacgagctg taccgtgcca ccatggccaa gcctttgtct caagaagaat
     3181 ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct gaagactaca
     3241 gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc aatgtatatc
     3301 attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct gctgcggcag
     3361 ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct
     3421 gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg
     3481 acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt
     3541 gggagggcta aggtacaagt ccggactcag atccagcgct gataacccag cctccaccac
     3601 aaacaaggat gccggtaccg gccgataatc aacctctgga ttacaaaatt tgtgaaagat
     3661 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     3721 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     3781 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     3841 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     3901 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     3961 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     4021 aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
     4081 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     4141 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     4201 gggccgcctc cccgcatcgg cccaccacca ccaccaccac atgtgaattg atccaagctt
     4261 atcgataggc ctaggcctat cgataggcct aggcctatcg ataggcctaa cacgagccat
     4321 agataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct
     4381 gtaggtttgg caagctagct gcagtaacgg ccattttgca aggcatggaa aaataccaaa
     4441 ccaagaatag agaagttcag atcaagggcg ggtacatgaa aatagctaac gttgggccaa
     4501 acaggatatc tgcggtgagc agtttcggcc ccggcccggg gccaagaaca gatggtcacc
     4561 gcagtttcgg ccccggcccg aggccaagaa cagatggtcc ccagatatgg cccaaccctc
     4621 agcagtttct taagacccat cagatgtttc caggctcccc caaggacctg aaatgaccct
     4681 gcgccttatt tgaattaacc aatcagcctg cttctcgctt ctgttcgcgc gcttctgctt
     4741 cccgagctct ataaaagagc tcacaacccc tcactcggcg cgccagtcct ccgattgact
     4801 gagtcgcccg ggtacccgta ttcccaataa agcctcttgc tgtttgcatc cgaatcgtgg
     4861 actcgctgat ccttgggagg gtctcctcag attgattgac tgcccacctc gggggtcttt
     4921 cagtaggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg
     4981 cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag
     5041 gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga
     5101 cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg
     5161 tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg
     5221 ccgacgcgct gggctacgtc ttgctggcgt tcgcgacgcg aggctggatg gccttcccca
     5281 ttatgattct tctcgcttcc ggcggcatcg ggatgcccgc gttgcaggcc atgctgtcca
     5341 ggcaggtaga tgacgaccat cagggacagc ttcaaggatc gctcgcggct cttaccagcc
     5401 taacttcgat cactggaccg ctgatcgtca cggcgattta tgccgcctcg gcgagcacat
     5461 ggaacgggtt ggcatggatt gtaggcgcct gatgcggtat tttctcctta cgcatctgtg
     5521 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     5581 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     5641 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     5701 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     5761 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     5821 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5881 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5941 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     6001 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     6061 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     6121 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     6181 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     6241 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     6301 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     6361 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     6421 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     6481 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6541 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6601 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6661 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6721 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6781 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6841 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6901 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6961 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     7021 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     7081 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     7141 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     7201 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     7261 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     7321 cgaactgaga tacctacagc gtgagcattg agaaagcgcc acgcttcccg aagggagaaa
     7381 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     7441 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     7501 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7561 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     7621 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     7681 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     7741 accgcctctc cccgcgcgtt ggccgattca ttaatgcagg tcgagggggg gcccggtcac
     7801 gattagtcca atttgttaaa gacaggatat caggtggtcc aggctctagt tttgactcaa
     7861 caatatcacc agctgaagcc tatagagtac gagccataga tagaataaaa gattttattt
     7921 agtctccaga aaaagggggg
//
