LOCUS       sequence-420432-        6505 bp    DNA     circular SYN 15-APR-2025
DEFINITION  This vector contains rTTA expressed under CMV promoter and gRNA 
            expressed under U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6505)
  AUTHORS   Nguyen et al.
  TITLE     Implantation of engineered adipocytes that outcompete tumors for 
            resources suppresses cancer progression
  JOURNAL   bioRxiv 2023.03.28.534564
REFERENCE   2  (bases 1 to 6505)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2023.03.28.534564"
COMMENT     Sequence Label: rTTA-gRNA-AAV
FEATURES             Location/Qualifiers
     source          1..6505
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..780
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     polyA_signal    793..1000
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     989..1006
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     1051..1067
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1052..1068
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1071..1659
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             1177..1188
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1586..1606
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    1715..1748
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1768..2478)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2046..2065)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2315..2333
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      2475..2484
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2491..2544)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(join(2554..2559,2560..2595,2596..2598,2599..2634,
                     2635..2637,2638..2673,2674..2679,2680..3297))
                     /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
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     promoter        complement(3321..3524)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3350..3370)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3525..3828
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_RNA        complement(3953..4028)
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     primer_bind     complement(4118..4138)
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     protein_bind    4142..4175
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     repeat_region   4418..4558
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4418..4547
                     /label=AAV2 ITR
     rep_origin      4633..5088
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4720..4739)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4930..4951
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5105..5124)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5224..5246
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5284..5302)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5370..5474
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5475..5543,5544..6335)
                     /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(5693..5712)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
      721 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      781 gcggcctcta gctccccagc atgcctgcta ttctcttccc aatcctcccc cttgctgtcc
      841 tgccccaccc caccccccag aatagaatga cacctactca gacaatgcga tgcaatttcc
      901 tcattttatt aggaaaggac agtgggagtg gcaccttcca gggtcaagga aggcacgggg
      961 gaggggcaaa caacagatgg ctggcaacta gaaggcacag tcgaggctga tcagcgagct
     1021 ctaggaattc catgtttttc taggtctcga tcgaggtcga cggtatcgat gcggggaggc
     1081 ggcccaaagg gagatccgac tcgtctgagg gcgaaggcga agacgcggaa gaggccgcag
     1141 agccggcagc aggccgcggg aaggaaggtc cgctggattg agggccgaag ggacgtagca
     1201 gaaggacgtc ccgcgcagaa tccaggtggc aacacaggcg agcagccaag gaaaggacga
     1261 tgatttcccc gacaacacca cggaattgtc agtgcccaac agccgagccc ctgtccagca
     1321 gcgggcaagg caggcggcga tgagttccgc cgtggcaata gggaggggga aagcgaaagt
     1381 cccggaaagg agctgacagg tggtggcaat gccccaacca gtgggggttg cgtcagcaaa
     1441 cacagtgcac accacgccac gttgcctgac aacgggccac aactcctcat aaagagacag
     1501 caaccaggat ttatacaagg aggagaaaat gaaagccata cgggaagcaa tagcatgata
     1561 caaaggcatt aaagcagcgt atccacatag cgtaaaagga gcaacatagt taagaatacc
     1621 agtcaatctt tcacaaattt tgtaatccag aggttgatta tcgataagct tgatatcgaa
     1681 ttgtacctag tggaaccgga acccttaaac atgtataact tcgtataatg tatgctatac
     1741 gaagttatta ggtccctcga cgaattctta cttgtacagc tcgtccatgc cgccggtgga
     1801 gtggcggccc tcggcgcgtt cgtactgttc cacgatggtg tagtcctcgt tgtgggaggt
     1861 gatgtccaac ttgatgttga cgttgtaggc gccgggcagc tgcacgggct tcttggcctt
     1921 gtaggtggtc ttgacctcag cgtcgtagtg gccgccgtcc ttcagcttca gcctctgctt
     1981 gatctcgccc ttcagggcgc cgtcctcggg gtacatccgc tcggaggagg cctcccagcc
     2041 catggtcttc ttctgcatta cggggccgtc ggaggggaag ttggtgccgc gcagcttcac
     2101 cttgtagatg aactcgccgt cctgcaggga ggagtcctgg gtcacggtca ccacgccgcc
     2161 gtcctcgaag ttcatcacgc gctcccactt gaagccctcg gggaaggaca gcttcaagta
     2221 gtcggggatg tcggcggggt gcttcacgta ggccttggag ccgtacatga actgagggga
     2281 caggatgtcc caggcgaagg gcagggggcc acccttggtc accttcagct tggcggtctg
     2341 ggtgccctcg taggggcggc cctcgccctc gccctcgatc tcgaactcgt ggccgttcac
     2401 ggagccctcc atgtgcacct tgaagcgcat gaactccttg atgatggcca tgttatcctc
     2461 ctcgcccttg ctcaccatgg tggcgaccgg tgggccagga ttctcctcga cgtcaccgca
     2521 tgttagcaga cttcctctgc cctctcccga tcccccgggg agcatgtcaa ggtcaaaatc
     2581 gtcaagagcg tcagcaggca gcatatcaag gtcaaagtcg tcaagggcat cggctgggag
     2641 catgtctaag tcaaaatcgt caagggcgtc ggtcggcccg ccgctttcgc actttagctg
     2701 tttctccagg ccacatatga ttagttccag gccgaaaagg aaggcaggtt cggctccctg
     2761 ccggtcgaac agctcaattg cttgtctcag aagtgggggc atagaatcgg tggtaggtgt
     2821 ctctctttcc tcttttgcta cttgatgctc ctgttcctcc aatacgcagc ccagtgtaaa
     2881 gtggcccacg gcggacagag cgtacagtgc gttctccagg gagaagcctt gctgacacag
     2941 gaacgcgagc tgattttcca gggtttcgta ctgtttctct gttgggcggg tgccgagatg
     3001 cactttagcc ccgtcgcgat gtgagaggag agcacagcgg tatgacttgg cgttgttccg
     3061 cagaaagtct tgccatgact cgccttccag ggggcagaag tgggtatgat gcctgtccag
     3121 catctcgatt ggcagggcat cgagcagggc ccgcttgttc ttcacgtgcc agtacagggt
     3181 aggctgctca actcccagct tttgagcgag tttccttgtc gtcaggcctt cgataccgac
     3241 accattgagt aattccagag ctccgtttat gactttgctc ttgtccagtc tagacatcgg
     3301 atctgacggt tcactaaacc agctctgctt atatagacct cccaccgtac acgcctaccg
     3361 cccatttgcg tcaatggggc ggagttgtta cgacattttg gaaagtcccg ttgattttgg
     3421 tgccaaaaca aactcccatt gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca
     3481 aaccgctatc cacgcccatt gatgtactgc caaaaccgca tcaccatggt aatagcgatg
     3541 actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta ctgggcataa
     3601 tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg catatgatac
     3661 acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg acgtcaatgg
     3721 aaagtcccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg
     3781 tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg gaactccata
     3841 tatgggctat gaactaatga ccccgtaatt gattactatt aataactaat gcatggcggt
     3901 aatacggtta tccacgcggc cgcctaatgg atcctagtac tcgagaaaaa aatctcgcca
     3961 acaagttgac gagataaaca cggcattttg ccttgttata gtagattctg tttccagagt
     4021 actataactt cccattctcg ctcggtgcct cgggccaaca aggtggttct ccaagggata
     4081 cttatagtct caaaacacac aattacttta cagttagggt gagtttcctt ttgtgctgtt
     4141 tataacttcg tataatgtat gctatacgaa gttatagaaa tccaagccta tcatgtaaaa
     4201 tgtagctagt attaaaaaga acagattatc tgtcttttat cgcacattaa gcctctatag
     4261 ttactaggaa atattatatg caaattaacc ggggcagggg agtagccgag cttctcccac
     4321 aagtccgtgc gagggggccg gcgcgggcct agagatggcg cgtcggatct ctagagcggg
     4381 cccggtacta accaaactgg atctctgctg cggccgcagg aacccctagt gatggagttg
     4441 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     4501 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     4561 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     4621 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4681 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
     4741 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4801 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     4861 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4921 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
     4981 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
     5041 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     5101 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     5161 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     5221 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     5281 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     5341 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5401 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5461 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5521 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5581 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5641 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5701 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5761 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5821 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5881 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5941 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     6001 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     6061 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6121 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6181 gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6241 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6301 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6361 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6421 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6481 ccgtagaaaa gatcaaagga tcttc
//
