LOCUS       sequence-13755-e        7142 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 7142)
  AUTHORS   Hsiao EC, Nguyen TD, Ng JK, Scott MJ, Chang WC, Zahed H, Conklin BR
  TITLE     Constitutive Gs activation using a single-construct 
            tetracycline-inducible expression system in embryonic stem cells and
            mice.
  JOURNAL   Stem Cell Res Ther. 2011 Mar 4. 2(2):11.
  PUBMED    21375737
REFERENCE   2  (bases 1 to 7142)
  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 Cell 
            Res Ther. 2011 Mar 4. 2(2):11."
COMMENT     Sequence Label: pcDNA3.2 GW delCMV
FEATURES             Location/Qualifiers
     source          1..7142
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     294..313
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        294..312
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    342..466
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(895..1200)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(997..1016)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1542..2201)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2116..2135
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2202..2304)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2482..2606)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2632..2673
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2713..2732)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2757..2805
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3007..3435
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3094..3113)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3304..3325
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3444..3464)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3449..3778
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3629..3764
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3691..3710
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3845..4639
                     /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(3899..3918)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4509..4528
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4815..4936
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4852..4871)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4906..4925
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4985..5001)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4985..5001)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4998..5020)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5009..5025
                     /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(5033..5039,5040..5057,5058..5063))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5078..5099
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5216..5233)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5387..5975)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5467..5486)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6146..6937,6938..7006))
                     /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     6769..6788
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7007..7111)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg
      301 actcactata gggagaccca agctggctag ttaagctatc aacaagtttg tacaaaaaag
      361 ctgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt ttgcataaaa
      421 aacagactac ataatactgt aaaacacaac atatccagtc actatgaatc aactacttag
      481 atggtattag tgacctgtag tcgaccgaca gccttccaaa tgttcttcgg gtgatgctgc
      541 caacttagtc gaccgacagc cttccaaatg ttcttctcaa acggaatcgt cgtatccagc
      601 ctactcgcta ttgtcctcaa tgccgtatta aatcataaaa agaaataaga aaaagaggtg
      661 cgagcctctt ttttgtgtga caaaataaaa acatctacct attcatatac gctagtgtca
      721 tagtcctgaa aatcatctgc atcaagaaca atttcacaac tcttatactt ttctcttaca
      781 agtcgttcgg cttcatctgg attttcagcc tctatactta ctaaacgtga taaagtttct
      841 gtaatttcta ctgtatcgac ctgcagactg gctgtgtata agggagcctg acatttatat
      901 tccccagaac atcaggttaa tggcgttttt gatgtcattt tcgcggtggc tgagatcagc
      961 cacttcttcc ccgataacgg agaccggcac actggccata tcggtggtca tcatgcgcca
     1021 gctttcatcc ccgatatgca ccaccgggta aagttcacgg gagactttat ctgacagcag
     1081 acgtgcactg gccaggggga tcaccatccg tcgcccgggc gtgtcaataa tatcactctg
     1141 tacatccaca aacagacgat aacggctctc tcttttatag gtgtaaacct taaactgcat
     1201 ttcaccagtc cctgttctcg tcagcaaaag agccgttcat ttcaataaac cgggcgacct
     1261 cagccatccc ttcctgattt tccgctttcc agcgttcggc acgcagacga cgggcttcat
     1321 tctgcatggt tgtgcttacc agaccggaga tattgacatc atatatgcct tgagcaactg
     1381 atagctgtcg ctgtcaactg tcactgtaat acgctgcttc atagcacacc tctttttgac
     1441 atacttcggg tatacatatc agtatatatt cttataccgc aaaaatcagc gcgcaaatac
     1501 gcatactgtt atctggcttt tagtaagccg gatccacgcg attacgcccc gccctgccac
     1561 tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc atcacagacg
     1621 gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt ataatatttg
     1681 cccatggtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa atcaaaactg
     1741 gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa ccctttaggg
     1801 aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg tagaaactgc
     1861 cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg ctcatggaaa
     1921 acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt cattgccata
     1981 cggaattccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc cggataaaac
     2041 ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg aacggtctgg
     2101 ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg
     2161 gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct
     2221 gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt atggtgaaag
     2281 ttggaacctc ttacgtgccg atcaacgtct cattttcgcc aaaagttggc ccagggcttc
     2341 ccggtatcaa cagggacacc aggatttatt tattctgcga agtgatcttc cgtcacaggt
     2401 atttattcgg cgcaaagtgc gtcgggtgat gctgccaact tagtcgacta caggtcacta
     2461 ataccatcta agtagttgat tcatagtgac tggatatgtt gtgttttaca gtattatgta
     2521 gtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt ttacgtttct
     2581 cgttcagctt tcttgtacaa agtggttgat ctagagggcc cgcggttcga aggtaagcct
     2641 atccctaacc ctctcctcgg tctcgattct acgcgtaccg gttagtaatg agtttaaacg
     2701 ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc
     2761 aataaaaaga cagaataaaa cgcacgggtg ttgggtcgtt tgttcataaa cgcggggttc
     2821 ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
     2881 gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag
     2941 ccaacgtcgg ggcggcaggc cctgccatag cagatctgcg cagctggggc tctagggggt
     3001 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3061 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3121 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg gggcatccct ttagggttcc
     3181 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3241 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3301 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     3361 atttataagg gattttgggg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3421 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     3481 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     3541 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3601 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     3661 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     3721 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     3781 gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt
     3841 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     3901 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     3961 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     4021 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     4081 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     4141 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     4201 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     4261 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     4321 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     4381 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     4441 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     4501 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     4561 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     4621 tcttgacgag ttcttctgag cgggactctg gggttcgcga aatgaccgac caagcgacgc
     4681 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     4741 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     4801 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     4861 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     4921 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     4981 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5041 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     5101 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5161 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5221 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5281 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5341 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5401 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5461 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5521 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5581 aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5641 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5701 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5761 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5821 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5881 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5941 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6001 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6061 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6121 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6181 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6241 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6301 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6361 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6421 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6481 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6541 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6601 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6661 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6721 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6781 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6841 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6901 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6961 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7021 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7081 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     7141 tc
//
