LOCUS       sequence-274506-        7118 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 7118)
  AUTHORS   Zafarana G, Avery SR, Avery K, Moore HD, Andrews PW
  TITLE     Specific knockdown of OCT4 in human embryonic stem cells by 
            inducible short hairpin RNA interference.
  JOURNAL   Stem Cells. 2009 Apr . 27(4):776-82.
  PUBMED    19350677
REFERENCE   2  (bases 1 to 7118)
  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: "Stem Cells.
            2009 Apr . 27(4):776-82."
COMMENT     Sequence Label: pCAGTetRnls
FEATURES             Location/Qualifiers
     source          1..7118
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    1..135
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        149..528
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          802..1818
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1742..1764
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1826..1845
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     1874..1890
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1875..1891
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1922..1942
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1943..2560
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="SRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVK
                     NKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRP
                     TEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTD
                     SMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(2013..2032)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     misc_feature    join(2616..3190,3191..3193,3194..3202)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2783..2800)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3010..3029
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3203..3802
                     /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(3203..3222)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3699..3719
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3883..3900)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3889..4113
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4160..4176)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4160..4176)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4173..4195)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4184..4200
                     /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(4208..4214,4215..4232,4233..4238))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4253..4274
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4333..4528
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4379..4514
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4534..4668
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4736..4753)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4907..5495)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4987..5006)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5666..6457,6458..6526))
                     /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     6289..6308
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
       61 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
      121 tatcatgtct ggatctgata tcatcgtcga cattgattat tgactagtta ttaatagtaa
      181 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
      241 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      301 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      361 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      421 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      481 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc
      541 cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta
      601 tttatttttt aattattttg tgcagcgatg ggggcggggg gggggcnnnc gccaggcggg
      661 gcggggcggg gcgaggggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag
      721 agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa
      781 aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc
      841 gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc
      901 gggcgggacg gcccttctcc tccgggctgt aattagcgct tggtttaatg acggcttgtt
      961 tcttttctgt ggctgcgtga aagccttgag gggctccggg agggcccttt gtgcgggggg
     1021 agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg
     1081 ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg
     1141 cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac
     1201 aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc
     1261 gggctgcaac cccccctgca cccccctccc cgagttgctg agcacggccc ggcttcgggt
     1321 gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag
     1381 gtgggggtgc cgggcggggc ggggccgcct cgggccgggg agggctcggg ggaggggcgc
     1441 ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga gccgcagcca ttgcctttta
     1501 tggtaatcgt gcgagagggc gcagggactt cctttgtccc aaatctgtgc ggagccgaaa
     1561 tctgggaggc gccgccgcac cccctctagc gggcgcgggg cgaagcggtg cggcgccggc
     1621 aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct
     1681 ctccagcctc ggggctgtcc gcggggggac ggctgccttc gggggggacg gggcagggcg
     1741 gggttcggct tctggcgtgt gaccggcggc tctagagcct ctgctaacca tgttcatgcc
     1801 ttcttctttt tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt
     1861 ggcaaagaat tcctcgaggt cgacggtatc gataagcttg atatcgaatt cgccagccat
     1921 gccaaaaaag aagaggaagg tatctagatt agataaaagt aaagtgatta acagcgcatt
     1981 agagctgctt aatgaggtcg gaatcgaagg tttaacaacc cgtaaactcg cccagaagct
     2041 aggtgtagag cagcctacat tgtattggca tgtaaaaaat aagcgggctt tgctcgacgc
     2101 cttagccatt gagatgttag ataggcacca tactcacttt tgccctttag aaggggaaag
     2161 ctggcaagat tttttacgta ataacgctaa aagttttaga tgtgctttac taagtcatcg
     2221 cgatggagca aaagtacatt taggtacacg gcctacagaa aaacagtatg aaactctcga
     2281 aaatcaatta gcctttttat gccaacaagg tttttcacta gagaatgcat tatatgcact
     2341 cagcgctgtg gggcatttta ctttaggttg cgtattggaa gatcaagagc atcaagtcgc
     2401 taaagaagaa agggaaacac ctactactga tagtatgccg ccattattac gacaagctat
     2461 cgaattattt gatcaccaag gtgcagagcc agccttctta ttcggccttg aattgatcat
     2521 atgcggatta gaaaaacaac ttaaatgtga aagtgggtct ccgcggtaag atatcgcggc
     2581 cgctcgataa gcttgatatc gaattccgcc ccccccccct ctccctcccc cccccctaac
     2641 gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc
     2701 accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg
     2761 agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg
     2821 aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc
     2881 aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa
     2941 gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa
     3001 agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta
     3061 ccccattgta tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg
     3121 aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca
     3181 cgatgataat atggccacaa ccatgaccga gtacaagccc acggtgcgcc tcgccacccg
     3241 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     3301 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     3361 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt
     3421 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     3481 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     3541 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga
     3601 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     3661 cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg
     3721 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
     3781 gacccgcaag cccggtgcct gacgcccgcc ccacgacccg cagcgcccga ccgaaaggag
     3841 cgcacgaccc catgcatcga tgatctagag ctcgctgatc agcctcgact gtgccttcta
     3901 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     3961 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     4021 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     4081 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga acctgcagcc
     4141 caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4201 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4261 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4321 gccagcggat ccgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     4381 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     4441 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     4501 cttttttgga ggcctaggct tttgcaaaaa gctaacttgt ttattgcagc ttataatggt
     4561 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4621 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatccg ctgcattaat
     4681 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4741 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4801 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4861 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4921 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4981 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5041 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5101 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5161 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5221 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5281 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5341 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5401 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5461 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5521 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5581 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5641 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5701 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5761 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5821 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5881 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5941 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6001 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6061 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6121 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6181 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6241 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6301 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6361 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6421 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6481 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6541 aatgaccgct ttggaaaaac aaagactgta tttcctggaa attaatgttt attcaataaa
     6601 ctgtgtattc agctatattc acatagtggt gaggctgaaa tgaggcggga agaggcggtt
     6661 ggggcttaat tatatcaatt tgggtggccc cacagcgcct ccaaggcgcc agtcctgttt
     6721 tgacaagttg cctctggaag cctctcttct ttttctccag agtaagcgga ggccaggggc
     6781 ccccggcctc tgcttaatac taaaaaaaac agctgttgtc atagtaatga ttgggtggaa
     6841 acattccagg cctgggtgga gaggcttttt gcttcctctt gcaaaaccac actgacattc
     6901 caggcctggg tggagaggct ttttgcttcc tcttgcaaaa ccacactgcc ctctggaggg
     6961 cagttgccta gcaactaatt aaaagaggat gtcgcacggc cagctgcggt cagttagtca
     7021 cttcctgctt aactgacttg acattttcta ttttaagagt cgggaggaaa attactgtgt
     7081 tggaggccct ccgccatctt ctgaagctga atcgaatt
//
