LOCUS       sequence-410128-        7369 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knockdown of alpha-SMA. Construct has inverted repeats to be used in
            Sleeping Beauty transposon system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7369)
  TITLE     Castro Lab Plasmids 2023
REFERENCE   2  (bases 1 to 7369)
  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"
COMMENT     Sequence Label: pT2/sh-alpha-SMA/GFP4_Seq1/clone1
FEATURES             Location/Qualifiers
     source          1..7369
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(77..96)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     287..308
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     571..593
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     585..602
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     586..602
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     612..631
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        612..630
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     LTR             1017..1533
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    1597..1938
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             2005..2421
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     2351..2373
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     2389..2405
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     ncRNA           2461..2582
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           2609..2623
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           2650..2776
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     primer_bind     complement(2809..2829)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2814..3143
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2994..3129
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(3150..3152,3153..3155,3156..3869)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3195..3216)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3456..3475)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3803..3824
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             3942..4456
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4618..4634
                     /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(4642..4648,4649..4666,4667..4672))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4687..4708
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5167..5187)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5167..5185)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5206..5222)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5206..5222)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    5230..5246
                     /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(5254..5260,5261..5278,5279..5284))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5299..5320
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5437..5454)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5608..6196)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5688..5707)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6367..7158,7159..7227))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAAKLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6990..7009
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7228..7332)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(7359..7369,1..445)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 agcggcgcat taagcgcggc gggagtggtg gttacgcgca gcgtgaccgc tacacttgcc
       61 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
      121 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
      181 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
      241 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
      301 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
      361 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
      421 aacaaaatat taacgcttac aatttccatt cgccattcag gctgcgcaac tgttgggaag
      481 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
      541 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
      601 gtgagcgcgc gtaatacgac tcactatagg gcgaattgga gctcggatcc ctatacagtt
      661 gaagtcggaa gtttacatac acttaagttg gagtcattaa aactcgtttt tcaactactc
      721 cacaaatttc ttgttaacaa acaatagttt tggcaagtca gttaggacat ctactttgtg
      781 catgacacaa gtcatttttc caacaattgt ttacagacag attatttcac ttataattca
      841 ctgtatcaca attccagtgg gtcagaagtt tacatacact aagttgactg tgcctttaaa
      901 cagcttggaa aattccagaa aatgatgtca tggctttaga agcttgatct gaagcctata
      961 gagtacgagc catagataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg
     1021 aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg
     1081 aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag agacagcaga
     1141 atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac
     1201 agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc agatgtttcc
     1261 agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc
     1321 ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct
     1381 cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtacccgtat tcccaataaa
     1441 gcctcttgct gtttgcatcc gaatcgtgga ctcgctgatc cttgggaggg tctcctcaga
     1501 ttgattgact gcccacctcg ggggtctttc atttggaggt tccaccgaga tttggagacc
     1561 cctgcccagg gaccaccgac ccccccgccg ggaggtaagc tggccagcgg tcgtttcgtg
     1621 tctgtctctg tctttgtgcg tgtttgtgcc ggcatctaat gtttgcgcct gcgtctgtac
     1681 tagttagcta actagctctg tatctggcgg acccgtggtg gaactgacga gttctgaaca
     1741 cccggccgca accctgggag acgtcccagg gactttgggg gccgtttttg tggcccgacc
     1801 tgaggaaggg agtcgatgtg gaatccgacc ccgtcaggat atgtggttct ggtaggagac
     1861 gagaacctaa aacagttccc gcctccgtct gaatttttgc tttcggtttg gaaccgaagc
     1921 cgcgcgtctt gtctgctgca gcgctgcagc atcgttctgt gttgtctctg tctgactgtg
     1981 tttctgtatt tgtctgaaaa ttagggccag actgttacca ctcccttaag tttgacctta
     2041 ggtcactgga aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga
     2101 cgttgggtta ccttctgctc tgcagaatgg ccaaccttta acgtcggatg gccgcgagac
     2161 ggcaccttta accgagacct catcacccag gttaagatca aggtcttttc acctggcccg
     2221 catggacacc cagaccaggt cccctacatc gtgacctggg aagccttggc ttttgacccc
     2281 cctccctggg tcaagccctt tgtacaccct aagcctccgc ctcctcttcc tccatccgcc
     2341 ccgtctctcc cccttgaacc tcctcgttcg accccgcctc gatcctccct ttatccagcc
     2401 ctcactcctt ctctaggcgc cggaattaga tctctcgact agggataaca gggtaattgt
     2461 ttgaatgagg cttcagtact ttacagaatc gttgcctgca catcttggaa acacttgctg
     2521 ggattacttc ttcaggttaa cccaacagaa ggctcgagaa ggtatattgc tgttgacagt
     2581 gagcggccta gcaacactga ttcaattagt gaagccacag atgtaattga atcagtgttg
     2641 ctaggctgcc tactgcctcg gaattcaagg ggctacttta ggagcaatta tcttgtttac
     2701 taaaactgaa taccttgcta tctctttgat acatttttac aaagctgaat taaaatggta
     2761 taaattaaat cacttttttc aattcgccag cacagtggtc gaccctgtgg aatgtgtgtc
     2821 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     2881 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     2941 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     3001 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     3061 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     3121 ttggaggcct aggcttttgc aaaaagctca tggtgagcaa gggcgaggag ctgttcaccg
     3181 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     3241 ctggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     3301 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     3361 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     3421 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     3481 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     3541 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     3601 tctatatcat ggccgacaag cagaagaacg gcatcaaggc gaacttcaag atccgccaca
     3661 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     3721 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     3781 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     3841 ctctcggcat ggacgagctg tacaagtaat gaattaatta agaattatca agcttatcga
     3901 taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac cccacctgta
     3961 ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaata cataactgag
     4021 aatagagaag ttcagatcaa ggttaggaac agagagacag cagaatatgg gccaaacagg
     4081 atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg tccccagatg
     4141 cggtcccgcc ctcagcagtt tctagagaac catcagatgt ttccagggtg ccccaaggac
     4201 ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg
     4261 cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg gcgcgccagt
     4321 cctccgatag actgcgtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca
     4381 tccgacttgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc
     4441 agcgggggtc tttcatgggt aacagtttct tgaagttgga gaacaacatt ctgagggtag
     4501 gagtcgaata ttaagtaatc ctgactcaat tagccactgt tttgaatcca catactccaa
     4561 tactcctgaa atagttcatt atggacagcg cagaaagagc tggggagaat tgtgaaattg
     4621 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     4681 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4741 gggaaacctg tcgtgccaga tccatggact tcactagtgc gcgcggccgc tctagatggc
     4801 cagatctagc ttgtggaagg ctactcgaaa tgtttgaccc aagttaaaca atttaaaggc
     4861 aatgctacca aatactaatt gagtgtatgt aaacttctga cccactggga atgtgatgaa
     4921 agaaataaaa gctgaaatga atcattctct ctactattat tctgatattt cacattctta
     4981 aaataaagtg gtgatcctaa ctgacctaag acagggaatt tttactagga ttaaatgtca
     5041 ggaattgtga aaaagtgagt ttaaatgtat ttggctaagg tgtatgtaaa cttccgactt
     5101 caactgtata gggatcctct agctagagtc gacctcgcgg gggggcccgg tacccagctt
     5161 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     5221 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     5281 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5341 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5401 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5461 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5521 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5581 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5641 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5701 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5761 cctgtccgcc tttctccctt cgggaaccgt ggcgctttct catagctcac gctgtaggta
     5821 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5881 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5941 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     6001 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     6061 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6121 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6181 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     6241 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6301 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6361 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6421 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6481 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     6541 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6601 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6661 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6721 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6781 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaacttgg ccgcagtgtt
     6841 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6901 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6961 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     7021 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     7081 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7141 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7201 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     7261 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     7321 aggggttccg cgcacatttc cccgaaaagt gccacctgac gcgccctgt
//
