LOCUS       sequence-405659-        7369 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 7369)
  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 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"; journalName: "Nat 
            Commun."; date: "2022-06-24"; volume: "13(1):3606. doi"; pages: " 
            10.1038/s41467-022-31340-1"
COMMENT     Sequence Label: pT2/shCol1a1/GFP4_Seq1.3
FEATURES             Location/Qualifiers
     source          1..7369
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(84..103)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     294..315
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     578..600
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     592..609
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     593..609
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     619..638
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        619..637
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     LTR             1024..1540
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    1604..1945
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             2012..2428
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     2358..2380
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     2396..2412
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     ncRNA           2468..2589
                     /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           2616..2630
                     /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           2657..2783
                     /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(2816..2836)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2821..3150
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3001..3136
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(3157..3159,3160..3162,3163..3876)
                     /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(3202..3223)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3463..3482)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3810..3831
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             3949..4463
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4625..4641
                     /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(4649..4655,4656..4673,4674..4679))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4694..4715
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5174..5194)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5174..5192)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5213..5229)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5213..5229)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    5237..5253
                     /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(5261..5267,5268..5285,5286..5291))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5306..5327
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5444..5461)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5615..6203)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5695..5714)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6374..7165,7166..7234))
                     /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     6997..7016
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7235..7339)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(7366..7369,1..452)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 gccctgtagc ggcgcattaa gcgcggcggg agtggtggtt acgcgcagcg tgaccgctac
       61 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
      121 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
      181 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
      241 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
      301 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
      361 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
      421 gaattttaac aaaatattaa cgcttacaat ttccattcgc cattcaggct gcgcaactgt
      481 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
      541 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
      601 acggccagtg agcgcgcgta atacgactca ctatagggcg aattggagct cggatcccta
      661 tacagttgaa gtcggaagtt tacatacact taagttggag tcattaaaac tcgtttttca
      721 actactccac aaatttcttg ttaacaaaca atagttttgg caagtcagtt aggacatcta
      781 ctttgtgcat gacacaagtc atttttccaa caattgttta cagacagatt atttcactta
      841 taattcactg tatcacaatt ccagtgggtc agaagtttac atacactaag ttgactgtgc
      901 ctttaaacag cttggaaaat tccagaaaat gatgtcatgg ctttagaagc ttgatctgaa
      961 gcctatagag tacgagccat agataaaata aaagatttta tttagtctcc agaaaaaggg
     1021 gggaatgaaa gaccccacct gtaggtttgg caagctagct taagtaacgc cattttgcaa
     1081 ggcatggaaa atacataact gagaatagag aagttcagat caaggttagg aacagagaga
     1141 cagcagaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc
     1201 caagaacaga tggtccccag atgcggtccc gccctcagca gtttctagag aaccatcaga
     1261 tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc
     1321 agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagcccac
     1381 aacccctcac tcggcgcgcc agtcctccga tagactgcgt cgcccgggta cccgtattcc
     1441 caataaagcc tcttgctgtt tgcatccgaa tcgtggactc gctgatcctt gggagggtct
     1501 cctcagattg attgactgcc cacctcgggg gtctttcatt tggaggttcc accgagattt
     1561 ggagacccct gcccagggac caccgacccc cccgccggga ggtaagctgg ccagcggtcg
     1621 tttcgtgtct gtctctgtct ttgtgcgtgt ttgtgccggc atctaatgtt tgcgcctgcg
     1681 tctgtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa ctgacgagtt
     1741 ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc gtttttgtgg
     1801 cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg tggttctggt
     1861 aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt cggtttggaa
     1921 ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt gtctctgtct
     1981 gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc ccttaagttt
     2041 gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg tagatgtcaa
     2101 gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg tcggatggcc
     2161 gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg tcttttcacc
     2221 tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag ccttggcttt
     2281 tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc ctcttcctcc
     2341 atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat cctcccttta
     2401 tccagccctc actccttctc taggcgccgg aattagatct ctcgactagg gataacaggg
     2461 taattgtttg aatgaggctt cagtacttta cagaatcgtt gcctgcacat cttggaaaca
     2521 cttgctggga ttacttcttc aggttaaccc aacagaaggc tcgagaaggt atattgctgt
     2581 tgacagtgag cggcaagaca gtcatcgaat acatagtgaa gccacagatg tatgtattcg
     2641 atgactgtct tgctgcctac tgcctcggaa ttcaaggggc tactttagga gcaattatct
     2701 tgtttactaa aactgaatac cttgctatct ctttgataca tttttacaaa gctgaattaa
     2761 aatggtataa attaaatcac ttttttcaat tcgccagcac agtggtcgac cctgtggaat
     2821 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     2881 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     2941 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3001 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3061 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     3121 ggcttttttg gaggcctagg cttttgcaaa aagctcatgg tgagcaaggg cgaggagctg
     3181 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     3241 agcgtgtctg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     3301 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     3361 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     3421 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     3481 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     3541 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     3601 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggcgaa cttcaagatc
     3661 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     3721 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     3781 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     3841 gggatcactc tcggcatgga cgagctgtac aagtaatgaa ttaattaaga attatcaagc
     3901 ttatcgataa aataaaagat tttatttagt ctccagaaaa aggggggaat gaaagacccc
     3961 acctgtaggt ttggcaagct agcttaagta acgccatttt gcaaggcatg gaaaatacat
     4021 aactgagaat agagaagttc agatcaaggt taggaacaga gagacagcag aatatgggcc
     4081 aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa cagatggtcc
     4141 ccagatgcgg tcccgccctc agcagtttct agagaaccat cagatgtttc cagggtgccc
     4201 caaggacctg aaatgaccct gtgccttatt tgaactaacc aatcagttcg cttctcgctt
     4261 ctgttcgcgc gcttctgctc cccgagctca ataaaagagc ccacaacccc tcactcggcg
     4321 cgccagtcct ccgatagact gcgtcgcccg ggtacccgtg tatccaataa accctcttgc
     4381 agttgcatcc gacttgtggt ctcgctgttc cttgggaggg tctcctctga gtgattgact
     4441 acccgtcagc gggggtcttt catgggtaac agtttcttga agttggagaa caacattctg
     4501 agggtaggag tcgaatatta agtaatcctg actcaattag ccactgtttt gaatccacat
     4561 actccaatac tcctgaaata gttcattatg gacagcgcag aaagagctgg ggagaattgt
     4621 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4681 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4741 tccagtcggg aaacctgtcg tgccagatcc atggacttca ctagtgcgcg cggccgctct
     4801 agatggccag atctagcttg tggaaggcta ctcgaaatgt ttgacccaag ttaaacaatt
     4861 taaaggcaat gctaccaaat actaattgag tgtatgtaaa cttctgaccc actgggaatg
     4921 tgatgaaaga aataaaagct gaaatgaatc attctctcta ctattattct gatatttcac
     4981 attcttaaaa taaagtggtg atcctaactg acctaagaca gggaattttt actaggatta
     5041 aatgtcagga attgtgaaaa agtgagttta aatgtatttg gctaaggtgt atgtaaactt
     5101 ccgacttcaa ctgtataggg atcctctagc tagagtcgac ctcgcggggg ggcccggtac
     5161 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc
     5221 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5281 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5341 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5401 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5461 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5521 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5581 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5641 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5701 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5761 ccggatacct gtccgccttt ctcccttcgg gaaccgtggc gctttctcat agctcacgct
     5821 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5881 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5941 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6001 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6061 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6121 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6181 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6241 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6301 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6361 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6421 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6481 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6541 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6601 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6661 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6721 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6781 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aacttggccg
     6841 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6901 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6961 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     7021 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7081 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7141 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7201 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7261 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7321 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgc
//
