LOCUS       sequence-394260-        7366 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 7366)
  TITLE     Castro-Lowenstein Lab Plasmids 2023
REFERENCE   2  (bases 1 to 7366)
  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_Seq2/clone1
FEATURES             Location/Qualifiers
     source          1..7366
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      7..462
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(94..113)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     304..325
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     LTR             1034..1550
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    1614..1955
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             2022..2435
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     2365..2387
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     2403..2419
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     ncRNA           2475..2596
                     /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           2623..2637
                     /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           2664..2790
                     /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(2823..2843)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2828..3157
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3008..3143
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(3164..3166,3167..3169,3170..3883)
                     /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(3209..3230)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3470..3489)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3817..3838
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             3956..4470
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4632..4648
                     /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(4656..4662,4663..4680,4681..4686))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4701..4722
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5181..5201)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5181..5199)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5220..5236)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5220..5236)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    5244..5260
                     /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(5268..5274,5275..5292,5293..5298))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5313..5334
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5451..5468)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5622..6210)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5702..5721)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6381..7172,7173..7241))
                     /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     7004..7023
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7242..7346)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg agtggtggtt acgcgcagcg
       61 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
      121 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
      181 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
      241 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
      301 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
      361 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
      421 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattggagct
      661 cggatcccta tacagttgaa gtcggaagtt tacatacact taagttggag tcattaaaac
      721 tcgtttttca actactccac aaatttcttg ttaacaaaca atagttttgg caagtcagtt
      781 aggacatcta ctttgtgcat gacacaagtc atttttccaa caattgttta cagacagatt
      841 atttcactta taattcactg tatcacaatt ccagtgggtc agaagtttac atacactaag
      901 ttgactgtgc ctttaaacag cttggaaaat tccagaaaat gatgtcatgg ctttagaagc
      961 ttgatctgaa gcctatagag tacgagccat agataaaata aaagatttta tttagtctcc
     1021 agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct taagtaacgc
     1081 cattttgcaa ggcatggaaa atacataact gagaatagag aagttcagat caaggttagg
     1141 aacagagaga cagcagaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc
     1201 ggctcagggc caagaacaga tggtccccag atgcggtccc gccctcagca gtttctagag
     1261 aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa
     1321 ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa
     1381 aagagcccac aacccctcac tcggcgcgcc agtcctccga tagactgcgt cgcccgggta
     1441 cccgtattcc caataaagcc tcttgctgtt tgcatccgaa tcgtggactc gctgatcctt
     1501 gggagggtct cctcagattg attgactgcc cacctcgggg gtctttcatt tggaggttcc
     1561 accgagattt ggagacccct gcccagggac caccgacccc cccgccggga ggtaagctgg
     1621 ccagcggtcg tttcgtgtct gtctctgtct ttgtgcgtgt ttgtgccggc atctaatgtt
     1681 tgcgcctgcg tctgtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa
     1741 ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc
     1801 gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg
     1861 tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt
     1921 cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt
     1981 gtctctgtct gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc
     2041 ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg
     2101 tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg
     2161 tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg
     2221 tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag
     2281 ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc
     2341 ctcttcctcc agccccgtct ctcccccttg aacctcctcg ttcgaccccg cctcgatcct
     2401 ccctttatcc agccctcact ccttctctag gcgccggaat tagatctctc gactagggat
     2461 aacagggtaa ttgtttgaat gaggcttcag tactttacag aatcgttgcc tgcacatctt
     2521 ggaaacactt gctgggatta cttcttcagg ttaacccaac agaaggctcg agaaggtata
     2581 ttgctgttga cagtgagcgg gaaggatctc tatgctaaca tagtgaagcc acagatgtat
     2641 gttagcatag agatccttcc tgcctactgc ctcggaattc aaggggctac tttaggagca
     2701 attatcttgt ttactaaaac tgaatacctt gctatctctt tgatacattt ttacaaagct
     2761 gaattaaaat ggtataaatt aaatcacttt tttcaattcg ccagcacagt ggtcgaccct
     2821 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2881 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     2941 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     3001 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     3061 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     3121 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcatggtga gcaagggcga
     3181 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     3241 caagttcagc gtgtctggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     3301 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     3361 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     3421 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     3481 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     3541 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     3601 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggcgaactt
     3661 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     3721 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     3781 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     3841 cgccgccggg atcactctcg gcatggacga gctgtacaag taatgaatta attaagaatt
     3901 atcaagctta tcgataaaat aaaagatttt atttagtctc cagaaaaagg ggggaatgaa
     3961 agaccccacc tgtaggtttg gcaagctagc ttaagtaacg ccattttgca aggcatggaa
     4021 aatacataac tgagaataga gaagttcaga tcaaggttag gaacagagag acagcagaat
     4081 atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg ccaagaacag
     4141 atggtcccca gatgcggtcc cgccctcagc agtttctaga gaaccatcag atgtttccag
     4201 ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga actaaccaat cagttcgctt
     4261 ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata aaagagccca caacccctca
     4321 ctcggcgcgc cagtcctccg atagactgcg tcgcccgggt acccgtgtat ccaataaacc
     4381 ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt gggagggtct cctctgagtg
     4441 attgactacc cgtcagcggg ggtctttcat gggtaacagt ttcttgaagt tggagaacaa
     4501 cattctgagg gtaggagtcg aatattaagt aatcctgact caattagcca ctgttttgaa
     4561 tccacatact ccaatactcc tgaaatagtt cattatggac agcgcagaaa gagctgggga
     4621 gaattgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     4681 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     4741 cccgctttcc agtcgggaaa cctgtcgtgc cagatccatg gacttcacta gtgcgcgcgg
     4801 ccgctctaga tggccagatc tagcttgtgg aaggctactc gaaatgtttg acccaagtta
     4861 aacaatttaa aggcaatgct accaaatact aattgagtgt atgtaaactt ctgacccact
     4921 gggaatgtga tgaaagaaat aaaagctgaa atgaatcatt ctctctacta ttattctgat
     4981 atttcacatt cttaaaataa agtggtgatc ctaactgacc taagacaggg aatttttact
     5041 aggattaaat gtcaggaatt gtgaaaaagt gagtttaaat gtatttggct aaggtgtatg
     5101 taaacttccg acttcaactg tatagggatc ctctagctag agtcgacctc gcgggggggc
     5161 ccggtaccca gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg
     5221 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5281 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5341 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5401 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5461 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5521 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5581 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5641 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5701 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5761 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa ccgtggcgct ttctcatagc
     5821 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5881 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5941 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     6001 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6061 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6121 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6181 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6241 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6301 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6361 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6421 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6481 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6541 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6601 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6661 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6721 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6781 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaac
     6841 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6901 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6961 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     7021 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     7081 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     7141 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     7201 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     7261 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     7321 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgc
//
