LOCUS       sequence-394278-        7182 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knockdown of Collagen Type 1 alpha 1. 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 7182)
  AUTHORS   Comba A, Faisal SM, Dunn PJ, Argento AE, Hollon TC, Al-Holou WN, 
            Varela ML, Zamler DB, Quass GL, Apostolides PF, Abel C 2nd, Brown 
            CE, Kish PE, Kahana A, Kleer CG, Motsch S, Castro MG, Lowenstein PR
  TITLE     Spatiotemporal analysis of glioma heterogeneity reveals COL1A1 as an
            actionable target to disrupt tumor progression.
  JOURNAL   Nat Commun. 2022 Jun 24;13(1):3606. doi: 10.1038/s41467-022-31340-1.
  PUBMED    35750880
REFERENCE   2  (bases 1 to 7182)
  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: "Nat 
            Commun."; date: "2022-06-24"; volume: "13(1):3606. doi"; pages: " 
            10.1038/s41467-022-31340-1"
COMMENT     Sequence Label: pT2/shCol1a1/Scarlet-Seq1.1
FEATURES             Location/Qualifiers
     source          1..7182
                     /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..2438
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     2368..2390
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     2406..2422
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     ncRNA           2478..2599
                     /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           2627..2641
                     /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           2669..2795
                     /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"
     enhancer        2933..3236
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3237..3440
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3391..3411
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3479..3488
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3485..4180
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(3618..3636)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3886..3905
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    4181..4246
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    4370..4491
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4407..4426)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4461..4480
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4997..5017)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4997..5015)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5036..5052)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5036..5052)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5049..5071)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5060..5076
                     /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(5084..5090,5091..5108,5109..5114))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5129..5150
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5267..5284)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5438..6026)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5518..5537)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6197..6988,6989..7057))
                     /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     6820..6839
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7058..7162)
                     /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 atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat
     2401 cctcccttta tccagccctc actccttctc taggcgccgg aattagatct ctcgactagg
     2461 gataacaggg taattgtttg aatgaggctt cagtacttta cagaatcgtt gcctgcacat
     2521 cttggaaaca cttgctggga ttacttcttc aggttaaccc aacagaaggc tcgagaaggt
     2581 atattgctgt tgacagtgag cgccctggtg atactggtgt taaatagtga agccacagat
     2641 gtatttaaca ccagtatcac cagggtgcct actgcctcgg aattcaaggg gctactttag
     2701 gagcaattat cttgtttact aaaactgaat accttgctat ctctttgata catttttaca
     2761 aagctgaatt aaaatggtat aaattaaatc acttttttca attcgccagc acagtgggtt
     2821 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc attagttatt
     2881 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     2941 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     3001 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     3061 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     3121 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     3181 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
     3241 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
     3301 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
     3361 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
     3421 aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc taccggtcgc
     3481 caccatggtg agcaagggcg aggcagtgat caaggagttc atgcggttca aggtgcacat
     3541 ggagggctcc atgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     3601 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttctcctg
     3661 ggacatcctg tcccctcagt tcatgtacgg ctccagggcc ttcatcaagc accccgccga
     3721 catccccgac tactataagc agtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     3781 cttcgaggac ggcggcgccg tgaccgtgac ccaggacacc tccctggagg acggcaccct
     3841 gatctacaag gtgaagctcc gcggcaccaa cttccctcct gacggccccg taatgcagaa
     3901 gaagacaatg ggctgggaag cgtccaccga gcggttgtac cccgaggacg gcgtgctgaa
     3961 gggcgacatt aagatggccc tgcgcctgaa ggacggcggc cgctacctgg cggacttcaa
     4021 gaccacctac aaggccaaga agcccgtgca gatgcccggc gcctacaacg tcgaccgcaa
     4081 gttggacatc acctcccaca acgaggacta caccgtggtg gaacagtacg aacgctccga
     4141 gggccgccac tccaccggcg gcatggacga gctgtacaag tccggactca gatctcgagc
     4201 tcaagcttcg aattctgcag tcgacggtac cgcgggcccg ggatccaccg gatctagata
     4261 actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     4321 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     4381 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4441 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa
     4501 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     4561 ttaaccaata ggccgaaatc ggcaaccaca tccatggact tcactagtgc gcgcggccgc
     4621 tctagatggc cagatctagc ttgtggaagg ctactcgaaa tgtttgaccc aagttaaaca
     4681 atttaaaggc aatgctacca aatactaatt gagtgtatgt aaacttctga cccactggga
     4741 atgtgatgaa agaaataaaa gctgaaatga atcattctct ctactattat tctgatattt
     4801 cacattctta aaataaagtg gtgatcctaa ctgacctaag acagggaatt tttactagga
     4861 ttaaatgtca ggaattgtga aaaagtgagt ttaaatgtat ttggctaagg tgtatgtaaa
     4921 cttccgactt caactgtata gggatcctct agctagagtc gacctcgcgg gggggcccgg
     4981 tacccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat
     5041 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     5101 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     5161 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5221 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     5281 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5341 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5401 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5461 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5521 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5581 ttaccggata cctgtccgcc tttctccctt cgggaaccgt ggcgctttct catagctcac
     5641 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5701 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5761 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5821 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     5881 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5941 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6001 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6061 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6121 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6181 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6241 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6301 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6361 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6421 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6481 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6541 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6601 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaacttgg
     6661 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6721 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6781 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6841 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6901 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6961 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7021 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7081 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7141 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gc
//
