LOCUS       sequence-394275-        7180 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 7180)
  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 7180)
  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_Seq1.2
FEATURES             Location/Qualifiers
     source          1..7180
                     /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           2626..2640
                     /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           2667..2793
                     /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        2931..3234
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3235..3438
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3389..3409
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3477..3486
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3483..4178
                     /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
                     AVTVTQDASLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(3616..3634)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3884..3903
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    4179..4244
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    4368..4489
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4405..4424)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4459..4478
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4995..5015)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4995..5013)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5034..5050)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5034..5050)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5047..5069)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5058..5074
                     /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(5082..5088,5089..5106,5107..5112))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5127..5148
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5265..5282)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5436..6024)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5516..5535)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6195..6986,6987..7055))
                     /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     6818..6837
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7056..7160)
                     /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 cggcaacagt cgcttcacct acatagtgaa gccacagatg
     2641 tatgtaggtg aagcgactgt tgctgcctac tgcctcggaa ttcaaggggc tactttagga
     2701 gcaattatct tgtttactaa aactgaatac cttgctatct ctttgataca tttttacaaa
     2761 gctgaattaa aatggtataa attaaatcac ttttttcaat tcgccagcac agtgggttct
     2821 ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat tagttattaa
     2881 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     2941 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     3001 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     3061 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     3121 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     3181 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
     3241 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
     3301 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
     3361 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
     3421 gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta ccggtcgcca
     3481 ccatggtgag caagggcgag gcagtgatca aggagttcat gcggttcaag gtgcacatgg
     3541 agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     3601 agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttctcctggg
     3661 acatcctgtc ccctcagttc atgtacggct ccagggcctt catcaagcac cccgccgaca
     3721 tccccgacta ctataagcag tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     3781 tcgaggacgg cggcgccgtg accgtgaccc aggacgcctc cctggaggac ggcaccctga
     3841 tctacaaggt gaagctccgc ggcaccaact tccctcctga cggccccgta atgcagaaga
     3901 agacaatggg ctgggaagcg tccaccgagc ggttgtaccc cgaggacggc gtgctgaagg
     3961 gcgacattaa gatggccctg cgcctgaagg acggcggccg ctacctggcg gacttcaaga
     4021 ccacctacaa ggccaagaag cccgtgcaga tgcccggcgc ctacaacgtc gaccgcaagt
     4081 tggacatcac ctcccacaac gaggactaca ccgtggtgga acagtacgaa cgctccgagg
     4141 gccgccactc caccggcggc atggacgagc tgtacaagtc cggactcaga tctcgagctc
     4201 aagcttcgaa ttctgcagtc gacggtaccg cgggcccggg atccaccgga tctagataac
     4261 tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     4321 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     4381 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4441 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt
     4501 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     4561 aaccaatagg ccgaaatcgg caaccacatc catggacttc actagtgcgc gcggccgctc
     4621 tagatggcca gatctagctt gtggaaggct actcgaaatg tttgacccaa gttaaacaat
     4681 ttaaaggcaa tgctaccaaa tactaattga gtgtatgtaa acttctgacc cactgggaat
     4741 gtgatgaaag aaataaaagc tgaaatgaat cattctctct actattattc tgatatttca
     4801 cattcttaaa ataaagtggt gatcctaact gacctaagac agggaatttt tactaggatt
     4861 aaatgtcagg aattgtgaaa aagtgagttt aaatgtattt ggctaaggtg tatgtaaact
     4921 tccgacttca actgtatagg gatcctctag ctagagtcga cctcgcgggg gggcccggta
     4981 cccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag
     5041 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     5101 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     5161 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5221 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     5281 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5341 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5401 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5461 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5521 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5581 accggatacc tgtccgcctt tctcccttcg ggaaccgtgg cgctttctca tagctcacgc
     5641 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5701 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5761 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5821 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5881 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5941 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6001 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6061 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6121 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6181 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6241 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6301 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6361 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6421 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6481 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6541 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6601 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taacttggcc
     6661 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6721 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6781 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6841 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6901 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6961 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     7021 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     7081 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     7141 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
//
