LOCUS       sequence-308649-        7085 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7085)
  AUTHORS   Gupta PB, Kuperwasser C, Brunet JP, Ramaswamy S, Kuo WL, Gray JW, 
            Naber SP, Weinberg RA
  TITLE     The melanocyte differentiation program predisposes to metastasis 
            after neoplastic transformation.
  JOURNAL   Nat Genet. 2005 Oct . 37(10):1047-54.
  PUBMED    16142232
REFERENCE   2  (bases 1 to 7085)
  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: "Nat Genet. 
            2005 Oct . 37(10):1047-54."
COMMENT     Sequence Label: siSlug1
FEATURES             Location/Qualifiers
     source          1..7085
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        52..300
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     52..72
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     223..242
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    398..515
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        564..1074
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     993..1011
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1096..1695
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1096..1115)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1592..1612
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1823..2056
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2128..2249
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2165..2184)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2219..2238
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2289..2424
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2444..2463)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2445..2463)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2473..2490)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2473..2489)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2482..2504)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2631..3086
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2718..2737)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2928..2949
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3112..3216
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3217..3285,3286..4077)
                     /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(3435..3454)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4248..4836
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4737..4756
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4990..5007
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5124..5145
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5160..5165,5166..5183,5184..5190)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5198..5214
                     /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)."
     primer_bind     5203..5225
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5222..5238
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5222..5238
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5257..5277
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5259..5277
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5305..5531
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5532..5712
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5759..5884
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6377..6610
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6795..6839
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
ORIGIN
        1 tctcgacggt atcgatcacg agactagcct cgagcggccg cccccttcac cgagggccta
       61 tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga
      121 attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa
      181 tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc
      241 gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc
      301 ggtaactgga cacacataca gtgactcgag tcactgtatg tgtgtccagt ttttttgaat
      361 tctcgacctc gagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg
      421 gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag
      481 aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag
      541 atccactttg gccgcggctc gagggggttg gggttgcgcc ttttccaagg cagccctggg
      601 tttgcgcagg gacgcggctg ctctgggcgt ggttccggga aacgcagcgg cgccgaccct
      661 gggtctcgca cattcttcac gtccgttcgc agcgtcaccc ggatcttcgc cgctaccctt
      721 gtgggccccc cggcgacgct tcctgctccg cccctaagtc gggaaggttc cttgcggttc
      781 gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca ctagtaccct cgcagacgga
      841 cagcgccagg gagcaatggc agcgcgccga ccgcgatggg ctgtggccaa tagcggctgc
      901 tcagcagggc gcgccgagag cagcggccgg gaaggggcgg tgcgggaggc ggggtgtggg
      961 gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg cattctgcaa gcctccggag
     1021 cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg acctctctcc ccagggggat
     1081 ccaccggagc ttaccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     1141 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     1201 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     1261 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     1321 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     1381 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     1441 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag
     1501 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     1561 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     1621 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     1681 aagcccggtg cctgacgccc gccccacgac ccgcagcgcc cgaccgaaag gagcgcacga
     1741 ccccatgcat cggtaccttt aagaccaatg acttacaagg cagctgtaga tcttagccac
     1801 tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg
     1861 ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
     1921 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     1981 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     2041 tggaaaatct ctagcagtag tagttcatgt catcttatta ttcagtattt ataacttgca
     2101 aagaaatgaa tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa
     2161 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     2221 ggtttgtcca aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa
     2281 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     2341 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     2401 agtgaggagg cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta
     2461 ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     2521 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     2581 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg
     2641 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2701 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2761 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2821 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     2881 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     2941 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     3001 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     3061 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     3121 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3181 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     3241 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     3301 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     3361 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     3421 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     3481 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     3541 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     3601 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     3661 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     3721 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     3781 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     3841 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     3901 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     3961 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     4021 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     4081 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     4141 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     4201 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4261 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4321 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4381 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4441 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4501 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4561 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4621 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4681 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4741 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4801 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4861 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4921 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     4981 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     5041 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     5101 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     5161 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     5221 acaggaaaca gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa
     5281 caaaagctgg agctgcaagc ttaatgtagt cttatgcaat actcttgtag tcttgcaaca
     5341 tggtaacgat gagttagcaa catgccttac aaggagagaa aaagcaccgt gcatgccgat
     5401 tggtggaagt aaggtggtac gatcgtgcct tattaggaag gcaacagacg ggtctgacat
     5461 ggattggacg aaccactgaa ttgccgcatt gcagagatat tgtatttaag tgcctagctc
     5521 gatacataaa cgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     5581 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     5641 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     5701 aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct
     5761 ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact
     5821 ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag
     5881 cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg
     5941 gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt
     6001 cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct
     6061 acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac
     6121 cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat
     6181 agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg atcttcagac
     6241 ctggaggagg agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa
     6301 aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa
     6361 aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta
     6421 tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc
     6481 agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag
     6541 tctggggcat caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc
     6601 aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt
     6661 ggaatgctag ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg
     6721 agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc
     6781 aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt
     6841 ggaattggtt taacataaca aattggctgt ggtatataaa attattcata atgatagtag
     6901 gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc
     6961 agggatattc accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc
     7021 ccgaaggaat agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga
     7081 acgga
//
